Antitumor Activity of Noscapine in Combination with Doxorubicin in Triple Negative Breast Cancer

Background The aim of this study was to investigate the anticancer activity and mechanism of action of Noscapine alone and in combination with Doxorubicin against triple negative breast cancer (TNBC). Methods TNBC cells were pretreated with Noscapine or Doxorubicin or combination and combination index values were calculated using isobolographic method. Apoptosis was assessed by TUNEL staining. Female athymic Nu/nu mice were xenografted with MDA-MB-231 cells and the efficacy of Noscapine, Doxorubicin and combination was determined. Protein expression, immunohistochemical staining were evaluated in harvested tumor tissues. Results Noscapine inhibited growth of MDA-MB-231 and MDA-MB-468 cells with the IC50 values of 36.16±3.76 and 42.7±4.3 µM respectively. The CI values (<0.59) were suggestive of strong synergistic interaction between Noscapine and Doxorubicin and combination treatment showed significant increase in apoptotic cells. Noscapine showed dose dependent reduction in the tumor volumes at a dose of 150–550 mg/kg/day compared to controls. Noscapine (300 mg/kg), Doxorubicin (1.5 mg/kg) and combination treatment reduced tumor volume by 39.4±5.8, 34.2±5.7 and 82.9±4.5 percent respectively and showed decreased expression of NF-KB pathway proteins, VEGF, cell survival, and increased expression of apoptotic and growth inhibitory proteins compared to single-agent treatment and control groups. Conclusions Noscapine potentiated the anticancer activity of Doxorubicin in a synergistic manner against TNBC tumors via inactivation of NF-KB and anti-angiogenic pathways while stimulating apoptosis. These findings suggest potential benefit for use of oral Noscapine and Doxorubicin combination therapy for treatment of more aggressive TNBC.


Introduction
Approximately 30-40% of breast cancers are estrogen receptor (ER) negative and the triple negative breast cancer (TNBC i.e. negative for ERa, PR and Her2 amplification) are the most clinically aggressive breast tumors [1,2]. TNBC relapses quickly in response to clinical treatment as this subtype of breast cancer has a high histological grade and poor prognosis [3]. Patients with TNBC, which account for about 10-17% of all breast cancer cases [4], are often unresponsive to endocrine agents such as tamoxifen and less responsive to standard adjuvant therapy [5]. Specific targeted therapies are not available to improve clinical outcome among TNBC patients. [4,5] TNBC do not respond to endocrine agents or trastuzumab and can only be treated with chemotherapy and treatment options for these tumors are limited by frequent de novo or acquired resistance to chemotherapy [6]. The limited availability of current systemic treatment options for TNBC necessitates the search for newer chemotherapeutic regimens. A promising target for the treatment of these ER 2 breast tumors is the microtubule cytoskeleton [7]. The effectiveness of microtubuleinterfering agents, taxanes and vinca alkaloids in treatment of various cancers has been well studied [8]. However, the clinical utility of taxanes has been limited due to drug-resistance, need of i.v. infusion over a long period of time and associated toxicities [9,10]. This has prompted search for microtubule targeting agent that may be administered orally, display favorable toxicity profiles and have better therapeutic indices in the treatment of TNBC. Noscapine attenuates microtubule dynamics just enough to activate the mitotic checkpoints to stop cell cycle and does not alter the steady state monomer/polymer ratio of tubulin [11,12]. Noscapine showed antitumor activity against a variety of cancer types (melanoma [13], ovarian [14], lymphoma [15], human myelogenous leukemia [16], gliobastoma [17], lung, [18] and breast [19]) both in vitro and in vivo while exerting minimal adverse side effects. Furthermore, Noscapine also showed little or no toxicity to the kidney, heart, liver, bone marrow, spleen, or small intestine and did not inhibit primary humoral immune responses in mice. Previous studies demonstrated that oral administration of Noscapine at 120 mg/kg and 300 mg/kg showed significant reduction in tumor volume in MCF-7 [19] and MDA-MB-231 [20] xenografts in nude mice. However, the effectiveness of Noscapine in combination with other anticancer agents for treatment of TNBC has not been studied yet. At present, the lack of highly effective therapeutic targets for TNBC leaves standard chemotherapy, for example use of combination of anthracycline and taxane, however these agents are insufficiently efficacious [21]. Doxorubicin is an anthracycline drug which is used as a chemotherapeutic agent for patients with metastatic breast cancer and has shown overall response rates between 35 and 50% in patients with TNBC who have not previously received chemotherapy [22]. Despite its excellent anti-tumor activity, Doxorubicin has a relatively low therapeutic index and its clinical utility is limited due to acute and chronic toxicities such as myelosuppression, immunosupression and dose-cumulative cardiotoxicity [23]. Therefore, combination treatment with another highly effective novel non-toxic drug which can lower the dose of chemotherapeutic agents would be desirable.
Given the challenge in treating ER 2 breast tumors and its inherent poor prognosis, the use of Noscapine in combination Doxorubicin will have major clinical implications for the treatment of ER 2 breast cancer. Based on the individual activity of these agents and their distinct mechanisms of action, we hypothesize that Noscapine in combination with Doxorubicin will produce additive or synergistic cytotoxic effects in human TNBC in vitro and in vivo possibly by inactivation of NF-KB and also via antiangiogenic and apoptotic activity. The objectives of this study are (a) to examine the anticancer activity of Noscapine alone and combination with Doxorubicin against TNBC cells, and (b) evaluate the antitumor effect of Noscapine alone and combination in mice bearing MDA-MB-231 xenograft TNBC tumors and elucidates underlying mechanism of action.

Materials and Methods
Noscapine and Doxorubicin were purchased from Sigma Chemicals, St. Louis, MO, USA and Spectrum Chemicals USA. The human breast cancer cell lines MDA-MB-231 and MDA-MB-468 were obtained from American Type Culture Collection (Rockville, MD, USA). Cells were grown in DMEM:F12K medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum. The cell culture media contained antibiotic antimycotic solution of penicillin (5,000 U/ml), streptomycin (0.1 mg/ml), and neomycin (0.2 mg/ml). The cells were maintained at 37uC in the presence of 5% CO 2 in air. The cells were maintained at 37uC in the presence of 5% CO 2 . All other chemicals were either reagent or tissue culture grade.

Animals
Female Nu/Nu mice (six weeks old form Harlan, Indianapolis, IN) were grouped and housed (n = 8 per cage) in sterile microisolator caging unit supplied with autoclaved Tek-Fresh bedding. The animals were kept under controlled conditions of 12:12 hour light: dark cycle, 2262uC and 50615 percent relative humidity. The mice were fed (irradiated rodent chow Harlan Teklad) and autoclaved water ad libitum. The animals were housed at Florida A and M University in accordance with the standards of the Guide for the Care and Use of Laboratory Animals and the Association for Assessment and Accreditation of Laboratory Animal Care.

In-vitro cytotoxicity studies
The MDA-MB-231 or MDA-MB-468 TNBC cell lines were plated in 96-well micro titer plates, at a density of 1610 4 cells/well and allowed to incubate overnight and were treated with various dilutions of Noscapine made in cell growth medium (10 to 160 mM) from Noscapine stock solution in DMSO. The cells were incubated for 72 h at 3760.2uC in a 5% CO 2 -jacketed incubator. To study the interaction between Noscapine and Doxorubicin, the MDA-MB-231 or MDA-MB-468 cells were treated with various dilutions of Doxorubicin in the presence or absence of Noscapine at 10, 20 and 30 mM. The plates were incubated for 72 h at 3760.2uC in a 5 percent CO 2 -jacketed incubator. Cell viability in each treatment group was determined by crystal violet dye assay. Separate study was done to find out the IC 50 values of Noscapine and Doxorubicin for the MDA-MB-231 and MDA-MB-468 cells.

Data analysis for the combination treatments
The percentage of cell survival as a function of drug concentration was then plotted to determine the IC 50 value (the drug concentration needed to prevent cell proliferation by 50%) [18,24]. The interactions between Doxorubicin and Noscapine were evaluated by isobolographic analysis, a dose-oriented geometric method of assessing drug interactions [25]. For 50 percent toxicity, the combination index (CI) values were calculated based on the equation stated below.
Where, Dx1 = Dose of drug 1 to produce 50 percent cell kill alone; D1 = Dose of drug 1 to produce 50 percent cell kill in combination with D2; Dx2 = Dose of drug 2 to produce 50 percent cell kill alone; D2 = Dose of drug 2 to produce 50 percent cell kill in combination with D1; a = 0 for mutually exclusive or 1 for mutually non-exclusive modes of drug action.

Induction of apoptosis in MDA-MB-231 cells
To detect apoptotic cells, the ApoTag Red In Situ Apoptosis detection kit R (Chemicon R International, CA, USA) was used. MDA-MB-231 cells were plated at a density of 1610 6 cells/well in 6-well plates and incubated overnight. Cells were treated with Doxorubicin (0.4 mg/ml), or Noscapine (30 mM), or combination. Untreated cells were used as control. After 72 h, cells were fixed in 4% paraformaldehyde and mounted onto slides using Cytospin R (Shandon). Equilibration buffer was added to slides and incubated for 10 minutes followed by incubation in working strength TdT enzyme at 37uC for 1 hour. The slides were incubated in stop/ wash buffer for 10 minutes at room temperature. Working strength anti-digoxinenin conjugate (rhodamine) was added to each slide for 30-minute incubation at room temperature. The images on the slides were visualized with an Olympus BX40 fluorescent microscope equipped with a computer-controlled digital camera (DP71, Olympus Center Valley, PA, USA). To quantify the apoptotic cells from terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, 100 cells from 6 random microscopic fields were counted.

In-vivo antitumor effect against MDA-MB-231 tumors
The adherent MDA-MB-231 tumor cells were washed with PBS, harvested from confluent cultures by 5-minute exposure to 0.25 percent trypsin and 0.02 percent EDTA solution in an incubator. Trypsinization was stopped with medium containing 10 percent FBS. The cells were centrifuged at 500 g for 4 min at 4uC and the floating cells in the supernatant were discarded. The cell pellet was resuspended in medium containing 10 percent FBS and mixed thoroughly. Trypan blue staining was used to determine the number of viable cells. The resuspended cells were dilutions of 3610 6 cells/100 ml were prepared in cell growth medium. The 100 ml of cell suspension was injected subcutaneously into right flank area of each mouse [18]. The protocol for in-vivo experiments with nude mice was approved by the Animal Care and Use Committee, Florida A and M University, Tallahassee, FL. The mice were randomized into vehicle control and treatment groups (n = 8) when xenografts were palpable with a tumor size of approximately 50 mm 3 . The mice were treated with i) 160 ml of vehicle; ii) Noscapine (150 mg/kg/day); iii) Noscapine (300 mg/ kg/day); iv) Noscapine (450 mg/kg/day); v) Noscapine (550 mg/ kg/day); vi) Doxorubicin (1.5 mg/kg/week, i.v.), vii) Noscapine (300 mg/kg/day)+Doxorubicin (1.5 mg/kg/week. i.v.). To check for evidence of toxicity, the animals were weighed twice weekly. The tumor dimensions were measured using a linear caliper and tumor volume was calculated using following equation: Where, V = tumor volume a = largest diameter of tumor b = smallest diameter of tumor The mice were fed with food and water ad libitum. On day 38, all animals were sacrificed by exposure to a lethal dose of halothane in a desiccator. After dissection and removal of the tumor tissues, the tumors were washed in sterile PBS. For immunohistochemistry (IHC), and TUNEL assay procedures, some of the tumors were fixed in formalin while others were rapidly frozen in liquid nitrogen and stored in 280uC.

TUNEL assay of xenograft MDA-MB-231 tumors
Formalin-fixed tumor tissues harvested 38 days after tumor implantation were embedded in paraffin and sectioned (4-5 mm thick). DeadEnd TM Colorimetric Apoptosis Detection System (Promega, Madison, WI) was used to detect apoptosis in the tumor sections placed on slides according to the manufacturer's protocol. Briefly, the equilibration buffer was added to slides and incubated for 10 minutes followed by 10-minute incubation in 20 mg/ml proteinase K solution. The sections were washed in PBS and incubated with TdT enzyme at 37uC for 1 hour in a humidified chamber for incorporation of biotinylated nucleotides at the 39-OH ends of DNA. The slides were incubated in horseradish peroxidase-labeled streptavidin to bind the biotinylated nucleotides followed by detection with stable chromagen DAB. The images on the slides were visualized with an Olympus BX40 light microscope equipped with a computer-controlled digital camera (DP71, Olympus Center Valley, PA, USA). Three slides per group were stained and apoptotic cells were identified by dark brown cytoplasmic staining.

Immunohistochemistry for Cleaved Caspase 3 and VEGF Expression of MDA-MB-231 tumors
Tumor tissue sections prepared from formalin-fixed, paraffinembedded tumor tissues were used for IHC studies according to the protocol specified in the SignalStain TM Cleaved Caspase-3 (Asp 175) IHC kit (Cell Signaling, Beverly, MA). The section slides were washed in xylene and hydrated in different concentrations of alcohol. The slides were heated in sodium acetate solution at 95uC for 10 minutes for antigen retrieval. The slides were washed three times in PBS and incubated with the primary antibody against cleaved caspase-3 overnight at 4uC. Horseradish peroxidaseconjugated secondary antibody was applied to locate the primary antibody. The specimens were stained with Nova Red stain and counterstained with hematoxylin. The presence of brown staining was considered a positive identification for activated caspase-3. For VEGF staining, the tissue sections were washed, hydrated and processed for antigen retrieval as described above for cleaved caspase-3 staining. The samples were incubated overnight at 4uC with either 1:50 dilution of VEGF antibody incubated with biotinylated secondary antibody followed by streptavidin. The color was developed by exposing the peroxidase to a substratechromagen, which forms a brown reaction product. VEGF expression was identified by the brown cytoplasmic staining. The Olympus BX40 light microscope equipped with computercontrolled digital camera (DP71, Olympus Center Valley, PA, USA) was used to visualize the images on the slides.

CD31 expression and Assessment of Microvessel Density of MDA-MB-231 tumors
Paraffin-embedded tumor tissues were deparaffinized and blocked for peroxidase activity as described under methodology for IHC for VEGF Expression. After washing with PBS, the sections were pretreated in citrate buffer in a microwave oven for 20 min at 92-98uC. After two washes with PBS, specimens were incubated in 10 percent normal goat serum (Atlanta Biologicals, GA,USA) for 20 min to reduce the nonspecific antibody binding. Subsequently, the sections were then incubated with a 1:500 diluted mouse CD31 monoclonal antibody (Cell Signaling Tech, MA), which is recognized as an endothelial cell surface marker, at room temperature for 1 h, followed by a 30 min treatment with HRP Rabbit/Mouse (Santa Cruz Biotechnology, Santa Cruz, CA,USA). After three washes with PBS, the section was developed with diaminobenzidene-hydrogen peroxidase substrate, and lightly counterstained with hematoxylin. To calculate microvessel density (MVD), three most vascularised areas of the tumour ('hot spots') were selected and mean values obtained by counting vessels. A single microvessel was defined as a discrete cluster of cells positive for CD31 staining, with no requirement for the presence of a lumen. Microvessel counts were performed at 6400 (640 objective lens and 610 ocular lens; 0.74 mm 2 per field).

Statistics
One-way ANOVA followed by Tukey's Multiple Comparison Test was performed to determine the significance of differences among groups using GraphPad PRISM version 3.0 software (SanDiego, CA). Differences were considered significant in all experiments at P,0.01 (*, significantly different from untreated controls; ** , significantly different from Noscapine and Doxorubicin single treatments.  Table 1).

Cell proliferation inhibition by Noscapine and synergistic behavior in combination with Doxorubicin
Induction of apoptotic DNA fragmentation in MDA-MB-231 cells Anti-tumor effect of Noscapine (300 mg/kg/day) and Doxorubicin combination in MDA-MB-231 xenograft model The results (Fig. 2B) show that tumor volume significantly decreased after treatment with Doxorubicin (1.5 mg/kg/week i.v. bolus, P,0.01), Noscapine (300 mg/kg oral, P,0.01), or combination (P,0.001) compared to control. Tumor volume for the combination treatment averaged 361664 mm 3 compared with 16486172 mm 3 for Noscapine treatment or 1259699 mm 3 for Doxorubicin treatment on day 38 post tumor implantation. It is evident that combination treatment was most effective in inhibiting tumor growth compared to Doxorubicin or Noscapine treatments. Furthermore, we did not observe any weight loss or other signs of toxicity in the mice treated with combination or Noscapine or Doxorubicin (Fig. 2D).

Effects on angiogenic and cell survival proteins in MDA-MB-231 xenograft model
We compared expression of angiogenic and survival protein in tumor lysates from control and treated mice by western blotting analysis using b-actin as loading control (Fig. 4). Noscapine treatment significantly (P,0.001) decreased expression of VEGF (Fig. 4A), and survivin (Fig. 4B) proteins except Noscapine at a dose of 150 mg/kg (P.0.05) compared to control. Noscapine treatment at 300, 450 and 550 mg/kg/day showed 0.14, 0.26 and 0.28 Fold decreased in VEGF expression inregressed tumor compared to vehicle treated control group. Combination treatment decreased expression of VEGF protein expression significantly (**, P,0.001) to 0.45-fold compared to 0.14-fold with Micron bar = 100 mm. Cells were quantitated by counting 100 cells from 6 random microscopic fields. Data are expressed as mean+SD (N = 6). Oneway ANOVA followed by post Tukey test was used for statistical analysis to compare control and treated groups. * P,0.01; all treatments significantly different from control and ** P,0.01; significantly different from Noscapine and Doxorubicin single treatments. doi:10.1371/journal.pone.0017733.g001 Noscapine (*, P,0.01) and 0.20-fold with Doxorubicin (*, P,0.01) treatment, respectively of controls in regressed tumors (Fig. 4A). The expression of survivin protein were significantly decreased by 0.44 fold (*, P,0.01), 0.15 fold (*, P,0.05) and 0.08 fold (*, P,0.05) with combination, Doxorubicin and Noscapine treatment compared to control group respectively (Fig. 4B).

DNA fragmentation and expression of cleaved caspase-3 in MDA-MB-231 xenograft model
To further investigate the role of apoptosis, tumor sections were stained with TUNEL for detection of DNA and expression of cleaved caspase-3 (Fig. 5). Noscapine (150-550 mg/kg/day) treated regressed tumors showed DNA fragmentation (Fig. 5A and 5C) and widespread staining of activated cleaved caspase-3 expression compared to controls (Fig. 5B and 5D), indicating that Noscapine induced apoptosis in a dose dependent fashion of MDA-MB-231 breast cancer cells in-vivo. Single-agent treatment with either Noscapine or Doxorubicin induced DNA fragmentation (brown staining) that was further significantly (** P,0.001) increased by combination treatment. The combination treatment led to apoptosis in 6565 percent of the tumor cells, whereas Noscapine and Doxorubicin induced apoptosis in percent 2063 and 3263 percent of the tumor cells respectively (Fig. 5A and  5C). Doxorubicin, Noscapine, and combination induced caspase-3 expression in tumors which was significantly (P,0.01) different compared to control tumors (Fig. 5 B and 5D). Combination, Doxorubicin and Noscapine treatment showed 6864, 3362, and 2263 percent increased expression of cleaved caspase 3 in tumors tissues respectively compared to control group ( Fig. 5B  and 5D).

Inhibition of angiogenesis by combination in MDA-MB-231 tumors
The highest expression of VEGF was seen in tumor tissues harvested from untreated mice (Fig. 6A & 6C). Decreased VEGF staining was observed in tumors treated with Noscapine (150-550 mg/kg/day) in dose dependent manner and combination (0.44-fold) compared to tumors treated with Doxorubicin (0.21fold) or Noscapine 300 mg/kg/day (0.1-fold) alone ( Fig. 6A and  6C). CD31 + endothelial cells were identified using IHC technique in harvested tumor tissues and the results are shown in Fig. 6B and 6D. The staining of CD31 + in Noscapine treated groups were significantly decreased to 0.05, 0.09, 0.18, and 0.28fold at doses of 150, 300, 450, and 550 mg/kg/day compared to control group respectively. The staining of CD31 + in combination, Doxorubicin (1.5 mg/kg) and Noscapine (300 mg/kg) treated groups were significantly decreased to 0.40, 0.17, and 0.09-fold compared to control group. The average microvessel per field in groups treated with Noscapine, Doxorubicin and combination were found to decreased by 1062.6 (P,0.05), 17.663.5 (P,0.01), and 40.665.0 (P,0.001) respectively compared control group. Discussion TNBC has a more aggressive clinical course than other forms of breast cancer [1,4]. Traditionally, chemotherapy has been the mainstay of systemic treatment for TNBC since currently available endocrine and HER2-directed therapies are ineffective. [21] Among anticancer agents, antimicrotubules (taxanes and vinca alkaloids) constitute one of the most effective chemotherapeutic agents for treatment of breast cancers. [7] However, their clinical utility is limited due to the development of drug resistance and associated severe side effects. [9,10] Noscapine is a safer orally active antimicrotubule agent showed in-vitro and in-vivo antitumor activity against variety of cancers including tumors resistant to conventional antimicrotubular agents [13,14,15,16,17,18,19] and did not exhibit severe side effects that are commonly seen with many chemotherapeutic agents. [13,14] Doxorubicin is an anthracycline drug which has shown significant anticancer activity against TNBC; [22] however its clinical utility has been limited due to low therapeutic index and associated adverse side effects [23]. In the present study, we demonstrated that Noscapine in combination with Doxorubicin was effective in a synergistic manner in inhibition of tumor growth of TNBC both in vitro and in vivo. Our results indicate that the anticancer activity of Noscapine alone and in combination with Doxorubicin was mediated via inactivation of NF-KB, induction of apoptosis, and inhibition of angiogenesis.
To our knowledge, this is the first study that demonstrates effectiveness of Noscapine in combination with Dox against TNBC. In this study, we demonstrated that Noscapine inhibits MDA-MB-231 and MDA-MB-468 cells proliferation in-vitro with IC 50 value of 36.1663.76 and 42.764.3 mM respectively which was comparable with IC 50 observed with MCF-7 breast (IC 50 = 42.3 mM), HeLa (IC 50 = 25 mM), and thymocyte (IC 50 = 10 mM) cells. [19] The antiproliferative activity of Noscapine was found to vary with the type and sensitivity of cancer cells. We used MDA-MB-231 and MDA-MB-468 TNBC cells to ascertain interaction between Noscapine (sub IC 50 concentration) and Doxorubicin using isobolographic method. We selected the isobolographic analysis method since it has been widely used to evaluate the interaction between two antitumor drugs and provide both qualitative and quantitative measure of nature and extent of drug interaction. [26] In the present investigation, isobolographic analysis showed that Noscapine enhanced the cytotoxicity of Doxorubicin (CI values,0.59) in MDA-MB-231 and MDA-MB-468 cells in a synergistic manner (Table 1). We recently reported that the CI values of ,1.0 are indicative of synergistic interactions in A549 and H460 cells using DIM-C-pPhC 6 H 5 in combination with Docetaxel [25]. Similarly, our recently conducted studies showed that the interaction between Noscapine and Cisplatin was synergistic (CI,0.6) against non-small cell lung cancer H460 and A549 cells [27]. Also, Hiser et al demonstrated that Noscapine in combination  . Western blotting of tumor tissue lysates to determine expressions apoptosis-related proteins (A) expression of NF-kb, IKBa, P-IKBa, Bax, Bcl2, caspase 3, cleaved caspase 3, activated caspase 8 and activated caspase 9 proteins in tumor lysates by western blotting and (B) quantitation of apoptotic protein expression. Tumor tissue lysates harvested tumor tissues from control-untreated and treated groups were analyzed by western blotting for protein expressions. Lane 1 = control; Lane 2 = Noscapine 150 mg/kg/day; Lane 3 = Noscapine 300 mg/kg/day; Lane 4 = Noscapine 450 mg/kg/day; Lane 5 = Noscapine 550 mg/kg/day; Lane 6 = Doxorubicin 1.5 mg/kg i.v. bolus, q3d67 schedule; Lane 7 = Combination (Noscapine 300 mg/kg/day+Doxorubicin1.5 mg/kg i.v. bolus, q3d67 schedule). Similar results were observed in triplicate experiments. Protein expression levels (relative to b-actin) were determined. Mean 6 SE for three replicate determinations. One-way ANOVA followed by post Tukey test was used for statistical analysis. P,0.01 (*, significantly different from untreated controls; ** , significantly different from Noscapine and Doxorubicin single treatments). doi:10.1371/journal.pone.0017733.g003 with vincristine showed synergistic (CI,1) in vitro against acute lymphoblastic CCRF-CEM and acute myelogenous leukemia HL-60 cells. [28] Inhibition of cell proliferation and/or induction of apoptotic cell death are key mechanisms by which chemotherapeutic agents exert their action [29]. To study the possible mechanism involved in the anticancer activity of Noscapine and combination, we evaluated induction of apoptosis of MDA-MB-231 cells. The TUNEL assay results showed induction of apoptosis at Noscapine 30 mM alone and in combination with Doxorubicin at 0.4 mg/ml of compared to untreated cells which was evident from positive TUNEL staining and chromatin condensation (Fig. 1). Induction of apoptosis has been demonstrated following Noscapine treatment in various types of human cancer cells like ovarian [14], H460 lung, [18] Hela and mouse thymocytes [19]. Combination treatment showed significant (P,0.001) induction of apoptosis in a synergistic manner in compared to single agent (Fig. 2). Similar to our results, combination treatment of Noscapine (150 mg/kg by gavage once daily) and 60 Co radiation (single fraction -25 Gy) showed significant (P,0.01) decreased proliferation and increased apoptosis (TUNEL positive cells) of GL261 tumor cells compared to single agent treatment [30].
After establishing the efficacy of Noscapine alone and combination with Doxorubicin on TNBC cells in-vitro, we designed in-vivo experiments to test the efficacy of Noscapine in combination with Doxorubicin against MDA-MB-231 xenograft model in nude mice. Our previous studies have shown dose dependant tumor reduction following oral administration of Noscapine (300-550 mg/kg) against lung cancer [18]. Therefore, we evaluated Noscapine in-vivo antitumor efficacy at different dose levels ranging from 150 to 550 mg/kg/day against MDA-MB-231 xenograft model in mice administered by oral gavage. Our results demonstrated that Noscapine at 550 mg/kg (p,0.01) through oral gavage showed higher reduction in tumor volume in MDA-MB-231 xenograft model compared to lower doses used (Fig. 3A). Similarly, Aneja et al reported that oral Noscapine (300 mg/kg) was able to suppress breast cancer progression in a xenograft model (s.c. inoculated 10 6 MDA-MB-231 cells) by 66 percent compared to control treatment at 24 days post tumor inoculation [20]. We observed 29 percent reduction in the tumor volume following Noscapine 300 mg/kg/day treatment compared to control due to use of 3610 6 MDA-MB-231 cells for s.c. inoculation and assessment of in vivo efficacy at 38 days post tumor inoculation.
Previous reports have indicated that Noscapine (120 mg/kg/ day, intraperitoneally) was effective in regressing MCF-7 breast tumors but the cell lines used were different than in our study. [19] In another investigation, oral administration of Noscapine at a dose of 300 mg/kg/day showed a significant regression of melanoma tumors compared to untreated animals [13]. The dose dependant antitumor activity of Noscapine in TNBC xenograft model may be attributed to: a) short plasma half life and its availability at the tumor site; and b) extensive first-pass metabolism that reduces the oral bioavailability of Noscapine [31,32]. There are very few reports available in literature on oral Noscapine absorption and pharmacokinetic parameters and a detailed systematic study is desirable. Our future studies will focus on improving the tumor targeting of Noscapine, so that it is effective at lower doses.
We evaluated the in vivo antitumor efficacy of combination in MDA-MB-231 xenograft tumors in Nu/nu mice using sub therapeutic dose of Noscapine 300 mg/kg/day administered by oral gavage and Doxorubicin 1.5 mg/kg/week by i.v. injection. Our in vivo results demonstrate synergistic behavior of combination in murine MDA-MB-231 xenograft tumor model. Interestingly, Noscapine, Doxorubicin and combination treatment showed non-significant change in weight loss suggesting favorable toxicity profile of Noscapine and Doxorubicin (Fig. 3C). Combination treatment will be advantageous over conventional taxane based combination therapy in treatment of TNBC due to improved patient compliance by oral administration of Noscapine with minimal adverse side effects. Landen et al. showed that in vivo anticancer activity of Noscapine at 300 mg/kg was comparable to that of paclitaxel 25 mg/kg against murine B16LS9 xenograft melanoma model [13]. However, neither additive nor synergistic effect was observed with Noscapine and Paclitaxel combination. Studies in our laboratory also showed that Noscapine and Docetaxel combination was neither additive nor synergistic against NSCLC both in vitro and in vivo which suggests that there was possibly a competition for the same target (unpublished data). On the contrary, our recent investigation demonstrated that Noscapine enhanced the anticancer activity of Cisplatin in an additive to synergistic manner against H460 lung xenografts model [27].
Several studies have provided evidence that enhanced tumor growth inhibition of breast tumors can be achieved by combining Doxorubicin with other agents such as zoledronic acid [33], interleukin-2 [34], TGFb Inhibitor [35], tetrathiomolybdate [36] as opposed to treating with single agent. In vivo studies by Bandyopadhyay et al showed that the small TGFb Inhibitor, TGFb1 (1 mg/kg every alternative day) and Doxorubicin (4 mg/ kg or 8 mg/kg once per 7 days) combination reduced tumor growth and lung metastasis by inhibition of epithelial-mesenchymal transition in the 4T1 orthotopic xenograft model in comparison to single treatments [35] and these results are consistent with those observed in our study.
To elucidate the underlying mechanism of action of Noscapine and combination treatment, we have evaluated the expression of NF-kB signaling, apoptotic, angiogenic and cell survival proteins using western blot. NF-kB mediates survival signals that inhibit apoptosis as well as promote cancer cell growth. [37] Recent reports indicate that Noscapine exerts inhibitory effect on NF-KB activation [38] and in our studies Noscapine and combination treatment inhibited NF-kB activation through inhibition of IKK activation, IkBa phosphorylation, and IkBa degradation in MDA-MB-231 xenograft TBNC tumors. Previous studies demonstrated that Noscapine induces multiple proapoptotic responses that induce apoptosis against variety of tumors [14,18,39,40,41,42]. Caspases are critical protease mediators of apoptosis triggered by different stimuli. [43] In the present study, we found that the Noscapine and combination treatment activated initiator caspases, such as caspase-8 and caspase-9 followed by activation of effector caspase-3. Results of our in vivo studies also demonstrate that Noscapine alone and combination treatment induced proapototic (Bax) or decreased Bcl 2 proteins (Fig. 3A and 3B) suggesting involvement of mitochondrial pathway. [39] Induction of apoptosis and expression of cleaved caspase 3 was significantly induced in vivo by combination treatment compared to Noscapine or Doxorubicin alone thus confirming that apoptosis is an important pathway associated with the anticancer activity of these compounds (Fig. 5A-D). Our recently published studies with Noscapine and Cisplatin combination also showed that their anticancer activity was mediated by induction of apoptosis via intrinsic and extrinsic pathways and inhibition of survival proteins in H460 lung tumors [27]. Ye et al demonstrated increased apoptotic activity in regressed tumor tissues following Noscapine treatment at 120 mg/kg/day against MCF-7 breast and Renal 1983 bladder tumor xenografts [19]. Our previous studies also showed induction of apoptosis and activation of cleaved caspase 3 following Noscapine treatment in the dose range of 300-550 mg/ kg/day in H460 xenografts [18]. Our in-vivo results showed increased apoptotic activity and correlated very well with our in-vitro results. Wang et al demonstrated that Zoledronic Acid and Doxorubicin combination therapy led to significant (P,0.05) increase in caspase-3-positive cells in MDA-G8 breast tumor xenografts compared to single agent therapy. [33] To gain more insights on the anticancer mechanisms of combination therapy, other non-apoptotic signaling pathways need to be investigated and these studies are in progress.
Angiogenesis is critical for establishing solid tumor growth and metastasis [44]. In this investigation, we observed that Noscapine and combination treatment significantly (p,0.001) decreased expression of VEGF (Fig. 4D) in regressed tumors compared to single agent treatment thereby suggesting inhibition of angiogenesis. In addition, Doxorubicin, Noscapine and combination treatment decreased expression of cell survival protein survivin which promotes angiogenesis in TNBC tumors ( Fig. 4A and 4B). Survivin was strongly upregulated in angiogenically stimulated endothelium in vitro and in vivo which protects endothelial cells from apoptosis. [44] The down regulation of survivin was correlated with down regulation of VEGF by Noscapine and combination treatment. Survivin also inhibits caspase activation and acts as a negative regulator [45]. Therefore, the downregulation of survivin expression results in activation of caspases and thereby induces apoptosis in tumor cells. Furthermore, our IHC results show that Noscapine and combination treatment decreased VEGF staining in tumor tissues harvested from mice compared to single agent treatment and control ( Fig. 6A and 6C). The tumor regression by Noscapine and combination was also mediated through decreased expression of VEGF and correlated very well with our VEGF expression results obtained with western blots of tumor lysates (Fig. 4D). MVD is a commonly used index of tumor angiogenic activity and we counted density of neovessels in histological sections of the tumor using CD31 staining. The CD31 expression (Fig. 6 B) and the average microvessels per field (Fig. 6D) in combination treated group were significantly (p,0.001) decreased compared to the single agent treated and control group. The decreased expression of angiogenic markers (IHC) and MVD in treatment compared to control groups appeared to correlate with our western blot results. Thus, Noscapine alone and in combination with Doxorubicin exhibited antiangiogenic activity, and the underlying mechanism is currently being further investigated in our laboratories.
In conclusion, our data provides compelling evidence that combination treatment is effective against MDA-MB-231 TNBC cells as well as tumor xenografts by inactivation of NF-kB, induction of apoptosis and inhibition of angiogenesis. While, the currently available chemotherapeutic agents are associated debilitating toxic side effects, oral Noscapine provides promise as an effective anticancer agent with significantly lower toxicity on normal cells. Thus the use of synergistically acting Noscapine and Doxorubicin combination therapy could be an innovative and promising therapeutic strategy for the treatment for TNBC and possibly will have fewer adverse side effects compared to currently available chemotherapeutic regimens.