j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 6 2 2 e6 3 0
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Antitumor activity of paclitaxel is significantly enhanced by a novel proapoptotic agent in nonesmall cell lung cancer Syed S. Razi, MD,a,b Sadiq Rehmani, MD,a,b Xiaogui Li, PhD,a,b Koji Park, MD,a,b Gary S. Schwartz, MD,a,b Mohammed J. Latif, MD,a,b and Faiz Y. Bhora, MDa,b,* a
Division of Thoracic Surgery, Department of Surgery, Mount Sinai St. Luke’s Hospital, Mount Sinai Health System, New York, NY, USA b Division of Thoracic Surgery, Department of Surgery, Mount Sinai Roosevelt Hospital, Mount Sinai Health System, New York, NY, USA
article info
abstract
Article history:
Background: Newer targeted agents are increasingly used in combination chemotherapy
Received 21 February 2014
regimens with enhanced survival and improved toxicity profile. Taxols, such as paclitaxel,
Received in revised form
independently potentiate tumor destruction via apoptosis and are used as first line therapy
26 July 2014
in patients with advanced nonesmall cell lung cancer (NSCLC). Procaspase-3-activating
Accepted 4 November 2014
compound-1 (PAC-1) is a novel proapoptotic agent that directly activates procaspase-3
Available online 10 November 2014
(PC-3) to caspase-3, leading to apoptosis in human lung adenocarcinoma cells. Hence, we sought to evaluate the antitumor effects of paclitaxel in combination with PAC-1.
Keywords:
Methods: Human NSCLC cell lines (A-549 and H-322m) were incubated in the presence of
Paclitaxel
PAC-1 and paclitaxel. Tumor cell viability was determined by a tetrazolium-based colori-
Procaspase-3 activating
metric assay (MTT assay). Western blot and flow cytometric analysis were performed to
compound-1 Nonesmall cell lung cancer
evaluate expression of PC-3 and the proportion of apoptotic cells, respectively. A xenograft murine model of NSCLC was used to study the in vivo antitumor effects of PAC-1. Results: PAC-1 significantly reduced the inhibitory concentration 50% of paclitaxel from 35.3 to 0.33 nM in A-549 and 8.2 to 1.16 nM in H-322m cell lines. Similarly, the apoptotic activity significantly increased to 85.38% and 70.36% in A-549 and H322m, respectively. Significantly enhanced conversion of PC-3 to caspase-3 was observed with PAC-1 paclitaxel combination (P < 0.05). Mice treated with a drug combination demonstrated 60% reduced tumor growth rate compared with those of controls (P < 0.05). Conclusions: PAC-1 significantly enhances the antitumor activity of paclitaxel against NSCLC. The activation of PC-3 and thus the apoptotic pathway is a potential strategy in the treatment of human lung cancer. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Icahn School of Medicine at Mount Sinai, 1000 10th Avenue, Ste 2B-07, New York, NY 10019. Tel.: þ1 212 523 7475; fax: þ1 212 523 8011. E-mail address: [email protected] (F.Y. Bhora). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.11.004
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 6 2 2 e6 3 0
1.
Background
Lung cancer remains the leading cause of cancer-related death in both men and women and is responsible for 1.3 million deaths worldwide annually [1]. Although surgical resection of early stage lung cancer provides the highest survival rates in patients with nonesmall lung cancer (NSCLC), many patients eventually develop progressive disease and require additional treatment [2]. During the last two decades, the introduction of molecular targeting agents has tremendously evolved the treatment paradigm of NSCLC. These molecular targeting agents are now being increasingly used as a personalized anticancer strategy [3e5]. Combination chemotherapy regimens, including cytotoxic chemotherapy and/or molecular targeting agents, may delay disease progression and prolong survival in advanced NSCLC, however with varying results [6,7]. Traditional cytotoxic agents, such as taxols, exert antiproliferative effects by inducing cell death in all rapidly dividing cell types, and are commonly used as the first line chemotherapy agent against NSCLC [8]. However, emerging evidence based on the underlying molecular mechanisms regulating cell-cycle suggest that rather than being intrinsically toxic, most anticancer drugs including paclitaxel merely stimulate tumor cells to self-destruct via apoptosis [9,10]. Essentially, these apoptotic pathways converge on the activation of executioner caspase(s) 3 and 7 [11]. Hence, procaspase-3-activating compounds (PACs) provide an attractive antitumor strategy by essentially bypassing the often redundant upstream pathways in lung cancer cells leading directly to apoptotic cell death. PAC-1 and the related analogs are small molecules that directly activate procaspase-3 (PC-3) through the chelation of inhibitory zinc ions [12,13]. As cancer cells have much higher levels of PC-3 compared with those of normal counterparts, PAC-1’s mechanism of action imparts a greater likelihood of success in inducing selective tumor cell death. PAC-1 has shown significant efficacy in inducing apoptotic cell death in various cultured cancer cell lines as well as in vivo murine and canine tumor models [12,14,15]. Hence, we sought to further characterize the efficacy of PAC-1 in combination with a known cytotoxic agent, paclitaxel. To our knowledge, this is the first study to evaluate the potential of targeted proapoptotic agents such as PAC-1 in a combination chemotherapy regimen.
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Eagle’s Medium (Invitrogen, Carlsbad, CA), supplemented with 10% heat inactivated fetal bovine serum, glutamine, penicillin (100 U/mL), and streptomycin (100 U/mL) in a humidified incubator containing 5% CO2 at 37 C.
2.2.
Viability assays
Cell viability was determined using the MTT assay (Invitrogen). A-549 and H-322m cells were plated at a concentration of 5 103 cells/200 mL and 1 104 cells/200 mL per well separately in 96-well plates (Fisher Scientific, Pittsburgh, PA). Three replicated wells were used for each assay. Six wells were used with only the culture medium to blank the spectrophotometer, and it was determined that the drug(s) alone or in combination did not influence the background color of the medium. The following day, cells were treated with fresh growth medium containing PAC-1 (Cayman Chemical, Ann Arbor, MI) at increasing concentrations of 0e20 mM in combination with paclitaxel (Bristol-Myers Squibb, New York, NY) or dimethyl sulfoxide (DMSO) (0.1%) for 72 h to achieve antiproliferative effects. After the treatment, 20 mM of MTT (freshly prepared 5 mg/mL in phosphate-buffered saline [PBS]) was added to each well and was kept in culture incubator for 4 h. After the careful removal of media, 150 mM of MTT solvent (4 mM HCl, 0.1% nondet P-40 [NP40] all in isopropranolol) was added. Optical density, which directly correlates to the viable cell proportion, was read at 590 nm with a reference filter of 620 nm.
2.3.
Flow cytometry
The cells were stained with annexin V-FITC and propidium iodide (PI) before flow cytometry as described in the apoptosis detection kit protocol (ApoScreen Annexin V Apoptosis Kit; Southern Biotech Company, Birmingham, AL). A-549 and H322m cell lines were plated at a concentration of 2 105 cells per well and 4 105 cells per well in six-well plates (Corning Cell Culture Plates; SigmaeAldrich, St. Louis, MO). Three replicated wells were used for each assay. The following day, cells were treated with fresh growth medium containing PAC1 and paclitaxel at varying dose concentrations for 24e36 h to achieve antiproliferative effects. All cells (adherent and nonadherent) were harvested by centrifugation and washed twice in PBS, stained with annexin V-FITC and PI, and flow cytometry performed to analyze apoptosis according to manufacturer’s instructions. Approximately 10,000 events (cells) were evaluated for each sample.
2.
Methods
2.4.
2.1.
Cell culture and reagents
Cultured cell protein extracts were prepared by cell pellets in cell lysis buffer (150-mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 50 mM Tris, pH 7.5) with protein inhibitor mix (Roche Diagnostics, Indianapolis, IN). Extracts were centrifuged at 12,000 rpm for 15 min at 4 C. Supernatant fractions were assayed for protein concentration by bicinchoninic acid assay with bovine serum albumin as reference standard (Thermo Scientific, Waltham, MA). Proteins were separated in a 4%e 20% Tris-Glycine gel (Invitrogen Corporation) and
We evaluated the effects of PAC-1 in two different human nonesmall cell lung adenocarcinoma cell lines. A-549 was acquired from American Type Culture Collection (Manassas, VA), and H-322m was acquired from National Cancer Institute (Frederick, MD). Both cell lines possess high levels of PC-3 and have been extensively studied in cancer chemotherapy against NSCLC [16,17]. Cell lines were cultured according to standard guidelines. Cells were grown in Dulbecco Modified
Western blot
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electrotransferred onto nitrocellulose membranes. The blots were blocked with TBST buffer (10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.05% Tween20) containing 5% non-fat dry milk and then incubated overnight at 4 C with mouse antie caspase-3 monoclonal antibody (T46 L) (Santa Cruz Biotechnology Inc, Dallas, TX) diluted in the same buffer. Subsequently, the blot was washed four times with TBST (without non-fat milk) and incubated for another 1 h with horseradish peroxidase-conjugated goat antiemouse secondary antibody (Santa Cruz Biotechnology Inc), which was diluted in TBST-5% non-fat milk. The membrane was again washed with TBST, and immunoreactive bands were detected by chemiluminescence (ECL kit, Invitrogen). The Western blot membranes were stripped and used for detecting b-actin band for the amount of protein as internal control by probing HRPconjugated mouse monoclonal actin antibodies (Santa Cruz Biotechnology Inc). Bands were analyzed by densitometry using ImageJ software (NIH, Bethesda, MD).
2.5.
Murine model of lung adenocarcinoma
NU/J female athymic nude mice (Jax Mice and Services, Bar Harbor, ME) between the ages of 3 and 5 wk were used for in vivo experiments. To eliminate potential gender bias, only female mice were used. The animals were housed in the animal care facilities at St. Luke’s Hospital and Roosevelt Hospital. A subcutaneous xenograft NSCLC model was used using the A-549 cell line. Mice were divided into the following treatment groups:
reached approximately 100 mm3, mice were randomized into different groups, and treatment initiated as per each designated group. PAC-1 was administered (from day 0) once daily via oral gavage in a mixture of 24:1 vegetable oil and/or DMSO for twenty-one consecutive days. Paclitaxel was administered intraperitoneally on day 0, once daily for 5 d. The control mice received vegetable oil and/or DMSO oral gavage and saline intraperitoneal injections. All mice were euthanized 1 wk after completion of treatment (day-28). Euthanasia was accomplished by 100% CO2 inhalation and necropsy performed with harvesting of the tumor mass in liquid nitrogen and storage at 80 C.
2.6. Multi polymerase chain reaction for caspase cascade of apoptosis A multi polymerase chain reaction (PCR) technique was used to analyze the differential messenger RNA expression levels of various caspase enzymes involved in apoptosis that is, caspase(s) 1, 2, 3, 8, 9, and apoptosis-activating factor-1 (Apaf-1). Briefly, we purified total RNA from the tumor specimens via TRIzol extraction method (Invitrogen Inc). Complementary DNA was synthesized using ThermoScript RT-PCR system kit (Invitrogen Inc). Caspase(s) 1, 2, 3, 8, 9, Apaf-1, and internal control 18S RNA gene products were amplified using the multi-PCR kit (Maxim Biotech Company, Rockville, MD). The PCR products were detected and expression levels compared by densitometry using ImageJ software.
2.7.
Group-A (Control) Group-B (100 mg kg1 PAC-1) Group-C (12 mg kg1 paclitaxel) Group-D (100 mg kg1 PAC-1 and 12 mg kg1 paclitaxel)
A total of 24 mice (n ¼ 6/group) were used for this study, averaging ages 7e8 wk at tumor inoculation and weighing approximately 25 g each. Normal power calculations based on the known variance of lung tumor volumes (from our previous experimentsedata not shown) in these mice illustrate that this number enabled us to detect a 20% difference among various groups (significance level 0.05, power 0.8, twotailed tests). All experiments were performed according to the standard guidelines established by the Institutional Animal Care and Use Committee at St. Luke’s Hospital and Roosevelt Hospital (protocol BH-0431). Tumor cell inoculums were prepared by first harvesting the cells in PBS buffer, resuspension in serum-free culture medium, counted in a hemocytometer, and equilibrated at a density of 2 106 cells per 100 mL. Tumor inoculation was performed under general anesthesia, using intraperitoneal administration of ketamine (100 mg kg1) þ xylazine (10 mg kg1). The tip of a 1.2 cm 27-gauge needle was advanced subcutaneously and tumor cells inoculated in the left-upper back region. Based on our preliminary experiments, the animals reliably developed subcutaneous tumors in 3e4 wk. The tumor volume was determined using the following formula: 0.5 a b2, where a is the largest diameter of the tumor and b is its perpendicular (assessed three times a week). When tumor size
Statistical analysis
Statistical analysis was performed using analysis of variance tests followed by post hoc Tukey tests or Student t-test as appropriate using SPSS software version 16.0 (SPSS Inc, Chicago, IL). Dose-response curves were generated using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA). Half-maximal inhibitory concentrations (IC50) were computed using nonlinear regression models after logarithmic dose transformations. Curve comparisons were performed using classic sigmoidal dose-response equations. Statistical significance was defined as P 0.05.
3.
Results
3.1.
PAC-1 enhances the cytotoxic effects of paclitaxel
We studied the antitumor effects of PAC-1 and paclitaxel alone and in combination. Dose-response curves were generated for these drugs using in vitro assays and halfmaximal IC50 computed. PAC-1 and paclitaxel drug combination was further evaluated in a xenograft mouse model of NSCLC. Treatment efficacy among different groups was analyzed by comparing the tumor volumes and expression levels of various caspase intermediates in harvested tumor specimens.
3.1.1.
PAC-1 significantly reduces the IC50 of paclitaxel
Cell viability was determined using MTT assay. IC50 was computed using nonlinear regression analysis. IC50 was
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Fig. 1 e MTT assay was used to study the dose-response curves of PAC-1 and paclitaxel in A549 (A) and H-322m (B) cell lines, after 72 h of treatment. IC50 was determined for both PAC-1 and paclitaxel alone and in combination. IC50 was computed using nonlinear regression model and statistical significance determined at P < 0.05. PAC-1 in combination with paclitaxel has shown significant antitumor effects against both A-549 and H-322m cell lines (P < 0.05). (Color version of the figure is available online.)
determined for both PAC-1 and paclitaxel. At 72 h, IC50 for PAC-1 was determined as 42 mM and 22 mM in A549 and H322m cell lines, respectively (data not shown). Similarly, IC50 for paclitaxel was determined to be 35.3 nM and 8.2 nM in A549 and H-322m cell lines, respectively. PAC-1 significantly enhanced the antitumor effects of paclitaxel and reduced the IC50 to 0.33 nM and 1.16 nM in A549 and H-322m cell lines, respectively (P < 0.05) as shown in Figure 1.
3.1.2. Tumor growth retardation by PAC-1 and paclitaxel in a murine model of NSCLC Athymic nude mice were treated with PAC-1 (100 mg kg1 per day) and paclitaxel (12 mg kg1 per day) as described previously; treatment dosages were adapted from the previous studies in murine models of NSCLC [12,18]. All three treatment groups demonstrated significant reduction of tumor growth rate as compared with that of controls (Fig. 2). The respective P values for group(s) B, C, and D were 0.05, 0.02, and 0.006. In particular, mice treated with PAC-1 and paclitaxel combination (group D) exhibited maximum tumor growth suppression, immediately after the initiation of treatment and the effects were sustained even after the treatment was completed. Overall, PAC-1 paclitaxel combination resulted in 60% reduced tumor growth (mean growth 425.9 63.1 mm3) compared with that of controls (mean growth 1077.4 400 mm3; P < 0.05). In all treatment groups, no significant side effects were observed.
3.2. PAC-1 paclitaxel combination regimen has significant proapoptotic effects Early phase of apoptosis is characterized by the loss of membrane phospholipid asymmetry, with translocation of phosphatidylserine from the inner leaflet of the phospholipid bilayer to the cell surface (as detected by annexin V binding
and PI counterstaining). A549 and H-322m cell lines were incubated with PAC-1 and paclitaxel alone, and in combination. PAC-1 significantly increased the proportion of apoptotic cells from 3.8% [ 0.33%] to 26.6% [ 1.6%] (P < 0.05) in A549 cell lines and 2.4% [ 0.06%] to 14.4% [ 1.08%] in H-322m cell lines (P < 0.05). Similarly, paclitaxel increased the level of apoptosis from 3.8% [ 0.33%] to 30% [ 1.5%] (P < 0.05) in A549 cell lines and 2.4% [ 0.06%] to 30% [ 1.0%] in H-322m cell lines (P < 0.05). However in combination, PAC-1 and paclitaxel demonstrated significantly enhanced proapoptotic effects and increased the percent apoptotic cell count to 85.38% [ 7.22%] in A-549 and 70.36% [ 0.73%] in H-322m cell lines (P < 0.05), as shown in Figure 3.
3.3. PAC-1 and paclitaxel upregulates the caspase cascade of apoptosis PC-3 molecule is a key regulator of caspase cascade and provides a powerful stimulus for the tumor cells to undergo apoptotic cell death. To evaluate whether PAC-1 and paclitaxel combination results in increased activation of PC-3 in human lung adenocarcinoma cell lines, we studied the expression levels in both cultured tumor cells and harvested tumor specimens.
3.3.1. Effects of PAC-1 and paclitaxel combination in the activation of PC-3 A-549 and H-322m cell lines were pretreated for 24 h with varying doses of PAC-1 and paclitaxel in vitro and expression of PC-3 levels analyzed using Western blots. Minimal activation of PC-3 was observed when tumor cells were pretreated with PAC-1 and paclitaxel alone at low doses. However, when these drugs were used in combination at similar doses, we observed a robust conversion of inactive zymogen PC-3 into active caspase-3, as shown in Figure 4.
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Fig. 2 e An in vivo subcutaneous model of human lung adenocarcinoma was established in nude mice using A-549 cell line. Paclitaxel was given intraperitoneally for five consecutive days at 12 mg/kg/d, whereas PAC-1 was administered orally at 100 mg/kg for 21 d. Tumor volumes were measured periodically to estimate growth rate. Tumor growth was significantly reduced in mice receiving both PAC-1 and paclitaxel (P [ 0.006). (Color version of the figure is available online.)
3.3.2. PAC-1 and paclitaxel significantly enhanced the expression of intermediate caspases
4.
Apaf-1, caspase-8, and caspase-3 are the key regulators of caspase cascade and are activated by varying stimuli in the intrinsic and extrinsic pathways of apoptosis. We studied the in vivo treatment effects of PAC-1 and paclitaxel combination on the expression of various mediators of caspase cascade (n ¼ 6 per group). A multi-PCR assay was used to analyze the expression levels of different caspases. PAC-1 significantly increased the expression levels of Apaf-1, caspase-3, and caspase-8 in vivo, as shown in Figure 5 (P < 0.05). PAC-1 and paclitaxel combination further enhanced the expression of these intermediate caspases; although the results were not statistically significant but an increasing trend was observed in all experiments.
Evasion of apoptosis is one of the classical hallmarks of cancer development and progression. A group of cysteine proteases called caspase(s) are the key regulators of apoptosis. This process involves a complex balance of several proapoptotic and antiapoptotic cellular proteins using over 150 known genes [11,19]. Hence, it is not surprising that cancer cells to survive must develop highly efficient means to counteract the various proapoptotic signals. These rapidly dividing tumor cells develop resistance by selecting the clones of cells that have lost the expression of genes crucial to the cell’s apoptotic cascade, and this process is further hastened by the inherent genomic instability of the cancer cells. Various proapoptotic
Discussion
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Fig. 3 e Flow cytometry was used to study the proapoptotic effects of PAC-1 and paclitaxel in A-549 and H-322m cell lines. Both drugs in combination produced significantly higher levels of apoptosis as shown in (A). Phosphatidylserine exposure (measured by annexin V and PI) as illustrated in R2/R3 gates (B) is significantly increased with PAC-1 and paclitaxel combination regimen in A-549 and H-322m cell lines after 24 and 36-h treatment, respectively. Pax [ paclitaxel; C-3 [ cleaved caspase-3. (Color version of the figure is available online.)
targeting agents have been developed that target specific proteins in the apoptotic cascade. In this study, we have demonstrated the proapoptotic effects of PAC-1 in lung cancer. In addition, we have also shown that PAC-1 significantly enhances the antitumor activity of paclitaxel, both in vitro and in NSCLC xenograft model. Our data on NSCLC complement previous studies on proapoptotic effects of PAC-1 in different cancers [20]. The proapoptotic effects of PAC-1 are proportional to the cellular levels of PC-3. Although PC-3 is present in both cancer cells and normal cellular counterparts, PAC-1 selectively induces apoptosis in cancer cells with high levels of PC-3. Reasons for this paradoxical elevation of PC-3 levels in various cancers are not fully understood; however, it is postulated that ineffective signal transduction confers tumor cell resistance to natural apoptotic signals with resultant elevation of cellular PC-3 levels. Under physiological conditions, intracellular Znþ inhibits the catalytic activity of PC-3. In vitro evidence suggests that by sequestering these divalent Znþ cations, PAC-1 essentially allows autocatalytic activation of PC-3 into caspase-3 resulting in apoptotic cell death [13,21]. In our study, PAC-1
administration led to a significant elevation of caspase-3 levels and increased proportion of apoptotic cells in both A549 and H-322m cell lines. Various drugs designed to restore programmed cell death have shown potential in preclinical models and early clinical trials. These include small molecules that disrupt p53-MDM2 interactions, bind Bcl-2, inhibit XIAP proteins, promote apoptosome formation or TRAIL induced celldeath [22e28]. Many of these drug targets modulate apoptotic cascade at early or intermediate positions and hence, mutations in downstream mediators will render these drugs ineffective. Activating PC-3 is biologically a sound approach as it bypasses all the upstream aberrations involving both intrinsic and extrinsic pathways of apoptosis [19]. Having established the antitumor and proapoptotic effects of PAC-1, we sought to evaluate the antitumor activity of paclitaxel in combination with PAC-1. Paclitaxel primarily binds to tubulin within the microtubule assembly resulting in its hyperstabilization with consequent cellcycle arrest in rapidly dividing tumor cells at G2/M phase. Failure to proceed with mitosis triggers apoptosis. The
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Fig. 4 e Both cell lines were pretreated with PAC-1 and paclitaxel at varying dosages in vitro, and cell cultures were studied for activation of apoptotic pathways, 24 h after treatment initiation. Minimal activation of caspase-3 was noted when cells were treated with PAC-1 and paclitaxel alone. However, combination regimen produced significantly increased expression of caspase-3 levels in H-322m and A-549 cell lines (A and B) (P < 0.05). Densitometry with b-actin normalization of immunoblots for PC-3 (C and D). (Color version of the figure is available online.)
process involves posttranslational modification of Bcl-2 antiapoptotic proteins, increased expression of proapoptotic Bax, activation of p34, cyclin-dependent kinases, and local release of tumor necrosis factor from macrophages [9]. Though eventually all these converge on the activation of caspase-cascade leading to cellular apoptosis, lung cancers often develop resistance against paclitaxel by altering their activities [11,29,30]. Furthermore, increasing the dose of paclitaxel for therapeutic efficacy is limited by its toxicity profile. We have shown that PAC-1 potentiates cytotoxic effects of paclitaxel in both A549 and H-322m cell lines. We also demonstrated that PAC-1 and paclitaxel combination leads to significant activation of PC-3 and resultant increased
apoptotic activity. We observed that PAC-1, in addition to directly activating PC-3, also led to increased expression levels of various upstream caspases. Hence, we postulate that activation of these intermediate caspases by PAC-1 complements the cytotoxic effects of paclitaxel by a feed-forward approach. We have also shown that pretreating cells with PAC-1 led to a significant decrease in IC50 for paclitaxel. This is an important finding; as discussed earlier, administration of higher doses of paclitaxel increases the morbid side effects of the drug. However, combining PAC-1 with paclitaxel can potentially reduce the required dose of paclitaxel to achieve maximal antitumor efficacy. Our in vitro findings were confirmed in vivo using an NSCLC xenograft model. This particular model was selected as it
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Fig. 5 e (A) The expression of different intermediate apoptotic enzymes was evaluated from tumor specimens of all 4 groups of mice. The multi-PCR results show a significant increase in the apoptotic levels of Caspase-3, Apaf-1 and Caspase-8 in PAC-1 containing regimen as opposed to control and Paclitaxel alone. One representative experiment of two performed in duplicate is shown. (B) Bands are analyzed by densitometry and the ratio of caspase enzymes to positive controls are graphed after normalization with 18sRNA. Apaf-1 levels were significantly enhanced (*P
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Antitumor activity of paclitaxel is significantly enhanced by a novel proapoptotic agent in nonesmall cell lung cancer Syed S. Razi, MD,a,b Sadiq Rehmani, MD,a,b Xiaogui Li, PhD,a,b Koji Park, MD,a,b Gary S. Schwartz, MD,a,b Mohammed J. Latif, MD,a,b and Faiz Y. Bhora, MDa,b,* a
Division of Thoracic Surgery, Department of Surgery, Mount Sinai St. Luke’s Hospital, Mount Sinai Health System, New York, NY, USA b Division of Thoracic Surgery, Department of Surgery, Mount Sinai Roosevelt Hospital, Mount Sinai Health System, New York, NY, USA
article info
abstract
Article history:
Background: Newer targeted agents are increasingly used in combination chemotherapy
Received 21 February 2014
regimens with enhanced survival and improved toxicity profile. Taxols, such as paclitaxel,
Received in revised form
independently potentiate tumor destruction via apoptosis and are used as first line therapy
26 July 2014
in patients with advanced nonesmall cell lung cancer (NSCLC). Procaspase-3-activating
Accepted 4 November 2014
compound-1 (PAC-1) is a novel proapoptotic agent that directly activates procaspase-3
Available online 10 November 2014
(PC-3) to caspase-3, leading to apoptosis in human lung adenocarcinoma cells. Hence, we sought to evaluate the antitumor effects of paclitaxel in combination with PAC-1.
Keywords:
Methods: Human NSCLC cell lines (A-549 and H-322m) were incubated in the presence of
Paclitaxel
PAC-1 and paclitaxel. Tumor cell viability was determined by a tetrazolium-based colori-
Procaspase-3 activating
metric assay (MTT assay). Western blot and flow cytometric analysis were performed to
compound-1 Nonesmall cell lung cancer
evaluate expression of PC-3 and the proportion of apoptotic cells, respectively. A xenograft murine model of NSCLC was used to study the in vivo antitumor effects of PAC-1. Results: PAC-1 significantly reduced the inhibitory concentration 50% of paclitaxel from 35.3 to 0.33 nM in A-549 and 8.2 to 1.16 nM in H-322m cell lines. Similarly, the apoptotic activity significantly increased to 85.38% and 70.36% in A-549 and H322m, respectively. Significantly enhanced conversion of PC-3 to caspase-3 was observed with PAC-1 paclitaxel combination (P < 0.05). Mice treated with a drug combination demonstrated 60% reduced tumor growth rate compared with those of controls (P < 0.05). Conclusions: PAC-1 significantly enhances the antitumor activity of paclitaxel against NSCLC. The activation of PC-3 and thus the apoptotic pathway is a potential strategy in the treatment of human lung cancer. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Icahn School of Medicine at Mount Sinai, 1000 10th Avenue, Ste 2B-07, New York, NY 10019. Tel.: þ1 212 523 7475; fax: þ1 212 523 8011. E-mail address: [email protected] (F.Y. Bhora). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.11.004
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 6 2 2 e6 3 0
1.
Background
Lung cancer remains the leading cause of cancer-related death in both men and women and is responsible for 1.3 million deaths worldwide annually [1]. Although surgical resection of early stage lung cancer provides the highest survival rates in patients with nonesmall lung cancer (NSCLC), many patients eventually develop progressive disease and require additional treatment [2]. During the last two decades, the introduction of molecular targeting agents has tremendously evolved the treatment paradigm of NSCLC. These molecular targeting agents are now being increasingly used as a personalized anticancer strategy [3e5]. Combination chemotherapy regimens, including cytotoxic chemotherapy and/or molecular targeting agents, may delay disease progression and prolong survival in advanced NSCLC, however with varying results [6,7]. Traditional cytotoxic agents, such as taxols, exert antiproliferative effects by inducing cell death in all rapidly dividing cell types, and are commonly used as the first line chemotherapy agent against NSCLC [8]. However, emerging evidence based on the underlying molecular mechanisms regulating cell-cycle suggest that rather than being intrinsically toxic, most anticancer drugs including paclitaxel merely stimulate tumor cells to self-destruct via apoptosis [9,10]. Essentially, these apoptotic pathways converge on the activation of executioner caspase(s) 3 and 7 [11]. Hence, procaspase-3-activating compounds (PACs) provide an attractive antitumor strategy by essentially bypassing the often redundant upstream pathways in lung cancer cells leading directly to apoptotic cell death. PAC-1 and the related analogs are small molecules that directly activate procaspase-3 (PC-3) through the chelation of inhibitory zinc ions [12,13]. As cancer cells have much higher levels of PC-3 compared with those of normal counterparts, PAC-1’s mechanism of action imparts a greater likelihood of success in inducing selective tumor cell death. PAC-1 has shown significant efficacy in inducing apoptotic cell death in various cultured cancer cell lines as well as in vivo murine and canine tumor models [12,14,15]. Hence, we sought to further characterize the efficacy of PAC-1 in combination with a known cytotoxic agent, paclitaxel. To our knowledge, this is the first study to evaluate the potential of targeted proapoptotic agents such as PAC-1 in a combination chemotherapy regimen.
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Eagle’s Medium (Invitrogen, Carlsbad, CA), supplemented with 10% heat inactivated fetal bovine serum, glutamine, penicillin (100 U/mL), and streptomycin (100 U/mL) in a humidified incubator containing 5% CO2 at 37 C.
2.2.
Viability assays
Cell viability was determined using the MTT assay (Invitrogen). A-549 and H-322m cells were plated at a concentration of 5 103 cells/200 mL and 1 104 cells/200 mL per well separately in 96-well plates (Fisher Scientific, Pittsburgh, PA). Three replicated wells were used for each assay. Six wells were used with only the culture medium to blank the spectrophotometer, and it was determined that the drug(s) alone or in combination did not influence the background color of the medium. The following day, cells were treated with fresh growth medium containing PAC-1 (Cayman Chemical, Ann Arbor, MI) at increasing concentrations of 0e20 mM in combination with paclitaxel (Bristol-Myers Squibb, New York, NY) or dimethyl sulfoxide (DMSO) (0.1%) for 72 h to achieve antiproliferative effects. After the treatment, 20 mM of MTT (freshly prepared 5 mg/mL in phosphate-buffered saline [PBS]) was added to each well and was kept in culture incubator for 4 h. After the careful removal of media, 150 mM of MTT solvent (4 mM HCl, 0.1% nondet P-40 [NP40] all in isopropranolol) was added. Optical density, which directly correlates to the viable cell proportion, was read at 590 nm with a reference filter of 620 nm.
2.3.
Flow cytometry
The cells were stained with annexin V-FITC and propidium iodide (PI) before flow cytometry as described in the apoptosis detection kit protocol (ApoScreen Annexin V Apoptosis Kit; Southern Biotech Company, Birmingham, AL). A-549 and H322m cell lines were plated at a concentration of 2 105 cells per well and 4 105 cells per well in six-well plates (Corning Cell Culture Plates; SigmaeAldrich, St. Louis, MO). Three replicated wells were used for each assay. The following day, cells were treated with fresh growth medium containing PAC1 and paclitaxel at varying dose concentrations for 24e36 h to achieve antiproliferative effects. All cells (adherent and nonadherent) were harvested by centrifugation and washed twice in PBS, stained with annexin V-FITC and PI, and flow cytometry performed to analyze apoptosis according to manufacturer’s instructions. Approximately 10,000 events (cells) were evaluated for each sample.
2.
Methods
2.4.
2.1.
Cell culture and reagents
Cultured cell protein extracts were prepared by cell pellets in cell lysis buffer (150-mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 50 mM Tris, pH 7.5) with protein inhibitor mix (Roche Diagnostics, Indianapolis, IN). Extracts were centrifuged at 12,000 rpm for 15 min at 4 C. Supernatant fractions were assayed for protein concentration by bicinchoninic acid assay with bovine serum albumin as reference standard (Thermo Scientific, Waltham, MA). Proteins were separated in a 4%e 20% Tris-Glycine gel (Invitrogen Corporation) and
We evaluated the effects of PAC-1 in two different human nonesmall cell lung adenocarcinoma cell lines. A-549 was acquired from American Type Culture Collection (Manassas, VA), and H-322m was acquired from National Cancer Institute (Frederick, MD). Both cell lines possess high levels of PC-3 and have been extensively studied in cancer chemotherapy against NSCLC [16,17]. Cell lines were cultured according to standard guidelines. Cells were grown in Dulbecco Modified
Western blot
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electrotransferred onto nitrocellulose membranes. The blots were blocked with TBST buffer (10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.05% Tween20) containing 5% non-fat dry milk and then incubated overnight at 4 C with mouse antie caspase-3 monoclonal antibody (T46 L) (Santa Cruz Biotechnology Inc, Dallas, TX) diluted in the same buffer. Subsequently, the blot was washed four times with TBST (without non-fat milk) and incubated for another 1 h with horseradish peroxidase-conjugated goat antiemouse secondary antibody (Santa Cruz Biotechnology Inc), which was diluted in TBST-5% non-fat milk. The membrane was again washed with TBST, and immunoreactive bands were detected by chemiluminescence (ECL kit, Invitrogen). The Western blot membranes were stripped and used for detecting b-actin band for the amount of protein as internal control by probing HRPconjugated mouse monoclonal actin antibodies (Santa Cruz Biotechnology Inc). Bands were analyzed by densitometry using ImageJ software (NIH, Bethesda, MD).
2.5.
Murine model of lung adenocarcinoma
NU/J female athymic nude mice (Jax Mice and Services, Bar Harbor, ME) between the ages of 3 and 5 wk were used for in vivo experiments. To eliminate potential gender bias, only female mice were used. The animals were housed in the animal care facilities at St. Luke’s Hospital and Roosevelt Hospital. A subcutaneous xenograft NSCLC model was used using the A-549 cell line. Mice were divided into the following treatment groups:
reached approximately 100 mm3, mice were randomized into different groups, and treatment initiated as per each designated group. PAC-1 was administered (from day 0) once daily via oral gavage in a mixture of 24:1 vegetable oil and/or DMSO for twenty-one consecutive days. Paclitaxel was administered intraperitoneally on day 0, once daily for 5 d. The control mice received vegetable oil and/or DMSO oral gavage and saline intraperitoneal injections. All mice were euthanized 1 wk after completion of treatment (day-28). Euthanasia was accomplished by 100% CO2 inhalation and necropsy performed with harvesting of the tumor mass in liquid nitrogen and storage at 80 C.
2.6. Multi polymerase chain reaction for caspase cascade of apoptosis A multi polymerase chain reaction (PCR) technique was used to analyze the differential messenger RNA expression levels of various caspase enzymes involved in apoptosis that is, caspase(s) 1, 2, 3, 8, 9, and apoptosis-activating factor-1 (Apaf-1). Briefly, we purified total RNA from the tumor specimens via TRIzol extraction method (Invitrogen Inc). Complementary DNA was synthesized using ThermoScript RT-PCR system kit (Invitrogen Inc). Caspase(s) 1, 2, 3, 8, 9, Apaf-1, and internal control 18S RNA gene products were amplified using the multi-PCR kit (Maxim Biotech Company, Rockville, MD). The PCR products were detected and expression levels compared by densitometry using ImageJ software.
2.7.
Group-A (Control) Group-B (100 mg kg1 PAC-1) Group-C (12 mg kg1 paclitaxel) Group-D (100 mg kg1 PAC-1 and 12 mg kg1 paclitaxel)
A total of 24 mice (n ¼ 6/group) were used for this study, averaging ages 7e8 wk at tumor inoculation and weighing approximately 25 g each. Normal power calculations based on the known variance of lung tumor volumes (from our previous experimentsedata not shown) in these mice illustrate that this number enabled us to detect a 20% difference among various groups (significance level 0.05, power 0.8, twotailed tests). All experiments were performed according to the standard guidelines established by the Institutional Animal Care and Use Committee at St. Luke’s Hospital and Roosevelt Hospital (protocol BH-0431). Tumor cell inoculums were prepared by first harvesting the cells in PBS buffer, resuspension in serum-free culture medium, counted in a hemocytometer, and equilibrated at a density of 2 106 cells per 100 mL. Tumor inoculation was performed under general anesthesia, using intraperitoneal administration of ketamine (100 mg kg1) þ xylazine (10 mg kg1). The tip of a 1.2 cm 27-gauge needle was advanced subcutaneously and tumor cells inoculated in the left-upper back region. Based on our preliminary experiments, the animals reliably developed subcutaneous tumors in 3e4 wk. The tumor volume was determined using the following formula: 0.5 a b2, where a is the largest diameter of the tumor and b is its perpendicular (assessed three times a week). When tumor size
Statistical analysis
Statistical analysis was performed using analysis of variance tests followed by post hoc Tukey tests or Student t-test as appropriate using SPSS software version 16.0 (SPSS Inc, Chicago, IL). Dose-response curves were generated using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA). Half-maximal inhibitory concentrations (IC50) were computed using nonlinear regression models after logarithmic dose transformations. Curve comparisons were performed using classic sigmoidal dose-response equations. Statistical significance was defined as P 0.05.
3.
Results
3.1.
PAC-1 enhances the cytotoxic effects of paclitaxel
We studied the antitumor effects of PAC-1 and paclitaxel alone and in combination. Dose-response curves were generated for these drugs using in vitro assays and halfmaximal IC50 computed. PAC-1 and paclitaxel drug combination was further evaluated in a xenograft mouse model of NSCLC. Treatment efficacy among different groups was analyzed by comparing the tumor volumes and expression levels of various caspase intermediates in harvested tumor specimens.
3.1.1.
PAC-1 significantly reduces the IC50 of paclitaxel
Cell viability was determined using MTT assay. IC50 was computed using nonlinear regression analysis. IC50 was
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Fig. 1 e MTT assay was used to study the dose-response curves of PAC-1 and paclitaxel in A549 (A) and H-322m (B) cell lines, after 72 h of treatment. IC50 was determined for both PAC-1 and paclitaxel alone and in combination. IC50 was computed using nonlinear regression model and statistical significance determined at P < 0.05. PAC-1 in combination with paclitaxel has shown significant antitumor effects against both A-549 and H-322m cell lines (P < 0.05). (Color version of the figure is available online.)
determined for both PAC-1 and paclitaxel. At 72 h, IC50 for PAC-1 was determined as 42 mM and 22 mM in A549 and H322m cell lines, respectively (data not shown). Similarly, IC50 for paclitaxel was determined to be 35.3 nM and 8.2 nM in A549 and H-322m cell lines, respectively. PAC-1 significantly enhanced the antitumor effects of paclitaxel and reduced the IC50 to 0.33 nM and 1.16 nM in A549 and H-322m cell lines, respectively (P < 0.05) as shown in Figure 1.
3.1.2. Tumor growth retardation by PAC-1 and paclitaxel in a murine model of NSCLC Athymic nude mice were treated with PAC-1 (100 mg kg1 per day) and paclitaxel (12 mg kg1 per day) as described previously; treatment dosages were adapted from the previous studies in murine models of NSCLC [12,18]. All three treatment groups demonstrated significant reduction of tumor growth rate as compared with that of controls (Fig. 2). The respective P values for group(s) B, C, and D were 0.05, 0.02, and 0.006. In particular, mice treated with PAC-1 and paclitaxel combination (group D) exhibited maximum tumor growth suppression, immediately after the initiation of treatment and the effects were sustained even after the treatment was completed. Overall, PAC-1 paclitaxel combination resulted in 60% reduced tumor growth (mean growth 425.9 63.1 mm3) compared with that of controls (mean growth 1077.4 400 mm3; P < 0.05). In all treatment groups, no significant side effects were observed.
3.2. PAC-1 paclitaxel combination regimen has significant proapoptotic effects Early phase of apoptosis is characterized by the loss of membrane phospholipid asymmetry, with translocation of phosphatidylserine from the inner leaflet of the phospholipid bilayer to the cell surface (as detected by annexin V binding
and PI counterstaining). A549 and H-322m cell lines were incubated with PAC-1 and paclitaxel alone, and in combination. PAC-1 significantly increased the proportion of apoptotic cells from 3.8% [ 0.33%] to 26.6% [ 1.6%] (P < 0.05) in A549 cell lines and 2.4% [ 0.06%] to 14.4% [ 1.08%] in H-322m cell lines (P < 0.05). Similarly, paclitaxel increased the level of apoptosis from 3.8% [ 0.33%] to 30% [ 1.5%] (P < 0.05) in A549 cell lines and 2.4% [ 0.06%] to 30% [ 1.0%] in H-322m cell lines (P < 0.05). However in combination, PAC-1 and paclitaxel demonstrated significantly enhanced proapoptotic effects and increased the percent apoptotic cell count to 85.38% [ 7.22%] in A-549 and 70.36% [ 0.73%] in H-322m cell lines (P < 0.05), as shown in Figure 3.
3.3. PAC-1 and paclitaxel upregulates the caspase cascade of apoptosis PC-3 molecule is a key regulator of caspase cascade and provides a powerful stimulus for the tumor cells to undergo apoptotic cell death. To evaluate whether PAC-1 and paclitaxel combination results in increased activation of PC-3 in human lung adenocarcinoma cell lines, we studied the expression levels in both cultured tumor cells and harvested tumor specimens.
3.3.1. Effects of PAC-1 and paclitaxel combination in the activation of PC-3 A-549 and H-322m cell lines were pretreated for 24 h with varying doses of PAC-1 and paclitaxel in vitro and expression of PC-3 levels analyzed using Western blots. Minimal activation of PC-3 was observed when tumor cells were pretreated with PAC-1 and paclitaxel alone at low doses. However, when these drugs were used in combination at similar doses, we observed a robust conversion of inactive zymogen PC-3 into active caspase-3, as shown in Figure 4.
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Fig. 2 e An in vivo subcutaneous model of human lung adenocarcinoma was established in nude mice using A-549 cell line. Paclitaxel was given intraperitoneally for five consecutive days at 12 mg/kg/d, whereas PAC-1 was administered orally at 100 mg/kg for 21 d. Tumor volumes were measured periodically to estimate growth rate. Tumor growth was significantly reduced in mice receiving both PAC-1 and paclitaxel (P [ 0.006). (Color version of the figure is available online.)
3.3.2. PAC-1 and paclitaxel significantly enhanced the expression of intermediate caspases
4.
Apaf-1, caspase-8, and caspase-3 are the key regulators of caspase cascade and are activated by varying stimuli in the intrinsic and extrinsic pathways of apoptosis. We studied the in vivo treatment effects of PAC-1 and paclitaxel combination on the expression of various mediators of caspase cascade (n ¼ 6 per group). A multi-PCR assay was used to analyze the expression levels of different caspases. PAC-1 significantly increased the expression levels of Apaf-1, caspase-3, and caspase-8 in vivo, as shown in Figure 5 (P < 0.05). PAC-1 and paclitaxel combination further enhanced the expression of these intermediate caspases; although the results were not statistically significant but an increasing trend was observed in all experiments.
Evasion of apoptosis is one of the classical hallmarks of cancer development and progression. A group of cysteine proteases called caspase(s) are the key regulators of apoptosis. This process involves a complex balance of several proapoptotic and antiapoptotic cellular proteins using over 150 known genes [11,19]. Hence, it is not surprising that cancer cells to survive must develop highly efficient means to counteract the various proapoptotic signals. These rapidly dividing tumor cells develop resistance by selecting the clones of cells that have lost the expression of genes crucial to the cell’s apoptotic cascade, and this process is further hastened by the inherent genomic instability of the cancer cells. Various proapoptotic
Discussion
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Fig. 3 e Flow cytometry was used to study the proapoptotic effects of PAC-1 and paclitaxel in A-549 and H-322m cell lines. Both drugs in combination produced significantly higher levels of apoptosis as shown in (A). Phosphatidylserine exposure (measured by annexin V and PI) as illustrated in R2/R3 gates (B) is significantly increased with PAC-1 and paclitaxel combination regimen in A-549 and H-322m cell lines after 24 and 36-h treatment, respectively. Pax [ paclitaxel; C-3 [ cleaved caspase-3. (Color version of the figure is available online.)
targeting agents have been developed that target specific proteins in the apoptotic cascade. In this study, we have demonstrated the proapoptotic effects of PAC-1 in lung cancer. In addition, we have also shown that PAC-1 significantly enhances the antitumor activity of paclitaxel, both in vitro and in NSCLC xenograft model. Our data on NSCLC complement previous studies on proapoptotic effects of PAC-1 in different cancers [20]. The proapoptotic effects of PAC-1 are proportional to the cellular levels of PC-3. Although PC-3 is present in both cancer cells and normal cellular counterparts, PAC-1 selectively induces apoptosis in cancer cells with high levels of PC-3. Reasons for this paradoxical elevation of PC-3 levels in various cancers are not fully understood; however, it is postulated that ineffective signal transduction confers tumor cell resistance to natural apoptotic signals with resultant elevation of cellular PC-3 levels. Under physiological conditions, intracellular Znþ inhibits the catalytic activity of PC-3. In vitro evidence suggests that by sequestering these divalent Znþ cations, PAC-1 essentially allows autocatalytic activation of PC-3 into caspase-3 resulting in apoptotic cell death [13,21]. In our study, PAC-1
administration led to a significant elevation of caspase-3 levels and increased proportion of apoptotic cells in both A549 and H-322m cell lines. Various drugs designed to restore programmed cell death have shown potential in preclinical models and early clinical trials. These include small molecules that disrupt p53-MDM2 interactions, bind Bcl-2, inhibit XIAP proteins, promote apoptosome formation or TRAIL induced celldeath [22e28]. Many of these drug targets modulate apoptotic cascade at early or intermediate positions and hence, mutations in downstream mediators will render these drugs ineffective. Activating PC-3 is biologically a sound approach as it bypasses all the upstream aberrations involving both intrinsic and extrinsic pathways of apoptosis [19]. Having established the antitumor and proapoptotic effects of PAC-1, we sought to evaluate the antitumor activity of paclitaxel in combination with PAC-1. Paclitaxel primarily binds to tubulin within the microtubule assembly resulting in its hyperstabilization with consequent cellcycle arrest in rapidly dividing tumor cells at G2/M phase. Failure to proceed with mitosis triggers apoptosis. The
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Fig. 4 e Both cell lines were pretreated with PAC-1 and paclitaxel at varying dosages in vitro, and cell cultures were studied for activation of apoptotic pathways, 24 h after treatment initiation. Minimal activation of caspase-3 was noted when cells were treated with PAC-1 and paclitaxel alone. However, combination regimen produced significantly increased expression of caspase-3 levels in H-322m and A-549 cell lines (A and B) (P < 0.05). Densitometry with b-actin normalization of immunoblots for PC-3 (C and D). (Color version of the figure is available online.)
process involves posttranslational modification of Bcl-2 antiapoptotic proteins, increased expression of proapoptotic Bax, activation of p34, cyclin-dependent kinases, and local release of tumor necrosis factor from macrophages [9]. Though eventually all these converge on the activation of caspase-cascade leading to cellular apoptosis, lung cancers often develop resistance against paclitaxel by altering their activities [11,29,30]. Furthermore, increasing the dose of paclitaxel for therapeutic efficacy is limited by its toxicity profile. We have shown that PAC-1 potentiates cytotoxic effects of paclitaxel in both A549 and H-322m cell lines. We also demonstrated that PAC-1 and paclitaxel combination leads to significant activation of PC-3 and resultant increased
apoptotic activity. We observed that PAC-1, in addition to directly activating PC-3, also led to increased expression levels of various upstream caspases. Hence, we postulate that activation of these intermediate caspases by PAC-1 complements the cytotoxic effects of paclitaxel by a feed-forward approach. We have also shown that pretreating cells with PAC-1 led to a significant decrease in IC50 for paclitaxel. This is an important finding; as discussed earlier, administration of higher doses of paclitaxel increases the morbid side effects of the drug. However, combining PAC-1 with paclitaxel can potentially reduce the required dose of paclitaxel to achieve maximal antitumor efficacy. Our in vitro findings were confirmed in vivo using an NSCLC xenograft model. This particular model was selected as it
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Fig. 5 e (A) The expression of different intermediate apoptotic enzymes was evaluated from tumor specimens of all 4 groups of mice. The multi-PCR results show a significant increase in the apoptotic levels of Caspase-3, Apaf-1 and Caspase-8 in PAC-1 containing regimen as opposed to control and Paclitaxel alone. One representative experiment of two performed in duplicate is shown. (B) Bands are analyzed by densitometry and the ratio of caspase enzymes to positive controls are graphed after normalization with 18sRNA. Apaf-1 levels were significantly enhanced (*P
