Black Horehound (Ballota nigra Linn) Induces Apoptosis in Prostate Cancer Cells (PC-3) Through Intrinsic Signalling Cascade

Cancer is one of the most dreadful disease causing millions of death. It accounts for ~7.6 million deaths (~13% of all deaths) worldwide, which is expected to rise to 13.1 million by 2030.1 Prostate cancer is the most commonly diagnosed cancer among men. The disease varies widely in its clinical aggressiveness. In some patients, prostate cancer metastasis rapidly, killing the patient within a year of initial clinical presentation, whereas other patients may live for many years with localized disease without apparent metastases.2 Androgen and androgen receptor play a major role in prostate cancer. Steroid hormone androgens contribute to the initiation and promotion of multistage carcinogenesis through binding to hormone receptors.3 Androgen ablation has been the standard form of therapy for advanced prostate cancer for over 50 years. This therapy targets only androgen-dependent cells leaving the androgenindependent cells, so, a majority of patients will not respond to this therapy.4 Apoptosis or active cell death is a process whereby cells die in response to specific signals. It is a physiological process that is essential for normal tissue development and maintenance of hemostasis. Through this process, damaged, unattached, mutant and aged cells are eliminated.5 Imperfection in the apoptotic pathway will lead to the growth of tumors and the development of resistance to anticancer therapies. In fact, suppression of apoptosis is considered as one of the hallmark of cancers.6 Current strategy of apoptotic target drugs targets at extrinsic pathway proteins or intrinsic pathway proteins. Extrinsic and intrinsic pathways are the two most important signaling pathways mediating apoptosis. Extrinsic pathway is mediated by cell surface death receptor and intrinsic is initiated in mitochondria.7 For both the pathways, caspases are the regulatory protein which cleaves a variety of proteins essential for cell survival, such as cytoskeletal proteins and DNA repair proteins.8


INTRODUCTION
Cancer is one of the most dreadful disease causing millions of death. It accounts for ~7.6 million deaths (~13% of all deaths) worldwide, which is expected to rise to 13.1 million by 2030. 1 Prostate cancer is the most commonly diagnosed cancer among men. The disease varies widely in its clinical aggressiveness. In some patients, prostate cancer metastasis rapidly, killing the patient within a year of initial clinical presentation, whereas other patients may live for many years with localized disease without apparent metastases. 2 Androgen and androgen receptor play a major role in prostate cancer. Steroid hormone androgens contribute to the initiation and promotion of multistage carcinogenesis through binding to hormone receptors. 3 Androgen ablation has been the standard form of therapy for advanced prostate cancer for over 50 years. This therapy targets only androgen-dependent cells leaving the androgenindependent cells, so, a majority of patients will not respond to this therapy. 4 Apoptosis or active cell death is a process whereby cells die in response to specific signals. It is a physiological process that is essential for normal tissue development and maintenance of hemostasis. Through this process, damaged, unattached, mutant and aged cells are eliminated. 5 Imperfection in the apoptotic pathway will lead to the growth of tumors and the development of resistance to anticancer therapies. In fact, suppression of apoptosis is considered as one of the hallmark of cancers. 6 Current strategy of apoptotic target drugs targets at extrinsic pathway proteins or intrinsic pathway proteins. Extrinsic and intrinsic pathways are the two most important signaling pathways mediating apoptosis. Extrinsic pathway is mediated by cell surface death receptor and intrinsic is initiated in mitochondria. 7 For both the pathways, caspases are the regulatory protein which cleaves a variety of proteins essential for cell survival, such as cytoskeletal proteins and DNA repair proteins. 8 Intrinsic apoptotic pathway is mainly governed by Bcl-2 family proteins. This family contains both proand antiapoptotic proteins. The antiapoptotic protein Bcl-2 is an oncogene that contributes to neoplastic progression by enhancing tumor cell survival through inhibition of apoptosis. In prostate cancer, this Bcl-2 is overexpressed which leads to progression of metastatic prostate cancer through inhibition of apoptotic cell death. 9 This Bcl-2 overexpression also causes resistance to heat-shock stress, several chemotherapies and radiotherapy. 10 Although great advancements have been made in the treatment and control of cancer progression, significant deficiencies and space for improvement remain. A number of undesired side effects sometimes occur during chemotherapy. 11 Compared with synthetic compounds, natural products provide inherent larger-scale diversity and have been the major resource of bioactive agents for new drug discovery. 12 In research point of view, natural products are rapidly being utilized as source for drug discovery and development, because of its comparatively safe and low cost. Anticancer agents from natural source have a long history in folk medicine and it has been combined into traditional and allopathic medicine. Nowadays, approximately 28% of modern medicines are derived from medicinal plants. 13 There are several drugs derived from the plant origin are used in market to treat cancer which includes vinca alkaloids-vinblastine and vincristine, epipodophyllotoxins (etoposide, teniposide), taxanes (paclitaxel, docetaxel), and camptothecin derivatives (topotecan, irinotecan). 14 Medicinal plants are at interest to the field of novel drug development, as most of the drug industries depend on medicinal plants for the production of novel bioactive compounds.
Ballota nigra Linn (Black horehound) is a three-foot, perennial herb of the family Lamiaceae which grows in waste ground, hedgerows, woods and shady places, preferring nitrogen-rich, moist, rather loose soil. The plant is found in Mediterranean and central Asia and distributed all over the Europe and the eastern United States and it is commonly known as Black horehound in English. 15 The aerial parts of the plant have been reported to contain various chemical constituents such as flavonoids, diterpenoids, phenylpropanoid glycosides, volatile oil and fatty acid. The whole plant is reported to exhibit various pharmacological activities such as antioxidant activity, hypoglycemic effect, neurosedative effect, antibacterial, insecticidal andanticholinesterase activity. 16,17 Rigano et al. 18 reported the cytotoxicity of B. nigra against two human cancer cells, hepatoma HepG2 cell line and breast cancer MCF-7 cell line. However, the elucidation of B.nigra on prostate cancer has not been studied so far. Hence, the proposed study the ethanolic extract of B.nigra was an attempt whether the selected plant extract could inhibit intrinsic pathway in human prostate cancer cells.

Procurement and maintenance of PC-3Cell line
PC-3 cell lines were obtained from the National Centre for Cell Science (Pune, India). The cells were grown in T-25 culture flasks in RPMI medium supplemented with 10% FBS with 1% penicillin/streptomycin and 1% amphotericin B. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . On attaining the confluence, the cells were trypsinized and plated.

Cell viability Test
The cell viability assay was performed by MTT assay, which determines mitochondrial activity in living cells. Cells were seeded in a 96-well plate at a density of 5×10 4 cells/well and incubated for 24 h at 37°C, 5% CO 2 incubator. After attachment, cells were washed with PBS and then incubated with serum-free medium for 6-12 h. EA was dissolved in DMSO with different concentration EA (50, 100, 200 and 400µg/ml) and added to the cells. After the treatment period, 20 ml medium was removed and 20 ml of MTT was added and incubated for 30 min. Then, 100 ml of DMSO was added to solubilize the crystals and was kept in dark for 10 min. The intensity of color development was measured at 570 nm in ELISA reader. The cell viability was calculated as follows: Cell viability = absorbance of treated cells/absorbance of control cells ×100%. Cell lysate preparation PC-3cells were placed in a Petri dish of 100mm × 20mm at a density concentration of 1 × 10 4 cells/Petri dish and grown in RPMI medium, respectively, with 10% FBS. After 24 h (~70-80% confluent), both the cells were treated with DAM (10 and 100 mM/ml) for 24 h. At the end of the treatment, cells were washed once with ice-cold PBS and added 300 ml of ice cold RIPA buffer with protease inhibitors. The Petri dishes were placed on ice and left on a shaker rocking for 2 min. Cell lysates were collected into a 1.5-ml tube and centrifuged at 14,000× g for 10 min at 4°C. The supernatant was separated and the protein concentration of supernatants was determined.

Protein expression analysis
Preparation of cell lysate PC-3 cells were plated in a petri-dish of 100mm20mm at a density concentration of 1×10 6 cells/petri-dish and grown in RPMI medium, respectively, with 10% FBS. After 24 h (~70-80% confluent), both the cells were treated with B.nigra (200µg/ml) for 24 h. At the end of treatment, cells were washed once with ice-cold PBS and added 300 ml of ice-cold RIPA buffer with protease inhibitors. The petri-dishes were placed on ice and left on a shaker rocking for 2 min. Cell lysates were collected into a 1.5-ml tube and centrifuged at 14 000 g for 10 min at 4ºC. The supernatants were separated and the protein concentration of supernatants was determined.

Protein isolation and western blotting
For protein isolation, 100 mg of adipose tissue from control and experimental animals were used. To 100 mg of adipose tissue, 1 ml of buffer A (5 mM NaN3, 0.25 M sucrose, 10 mM NaHCO3) was added and homogenized and centrifuged at 1300 ×g at 4 °C for 10 min. The supernatant was separated and again centrifuged at 4 °C for 15 min at 12,000 ×g. The final supernatant was specimened as a total protein to assess the post-receptor insulin signaling molecules. The protein estimation was done based on the method of Lowry, et al. 19 By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gel), The lysate proteins (50 μg/lane) were separated and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories Inc) by electroblotting. 5% of non-fat dry milk was used to block the membranes and tagged with the primary antibodies (1:1000 dilutions). The membrane was subjected to repeated wash with TBS-T for three times and incubated with 1:5000 dilution of horseradish peroxidase-conjugated rabbit-anti-mouse or goat-antirabbit secondary antibodies (GeNei, Bangalore, India) for 1 h. After incubation, the membrane was again subjected to repeated wash with TBS and TBS-T three times. The protein bands were visualized using a sophisticated Chemiluminescence detection system (Thermo Fisher Scientific Inc., Waltham, MA, USA), the specific signals were found and protein bands were captured and quantified by Chemidoc and Quantity One image analysis system Bio-Rad Laboratories, CA respectively. Later, the membrane was incubated at 500C in stripping buffer (50 ml, containing 62.5 mM of Tris-HCl (pH 6.7), 1 g of SDS and 0.34 ml of β-mercaptoethanol) for 30 min. Then, the membranes were reprobed with β-actin antibody (1:5000). β-actin was used as invariant control.

STATISTICAL ANALYSIS
Data were expressed as mean ± standard error of the mean. Statistical analyses were performed using one-way ANOVA followed by Duncan's tests for comparison between treatment and control values using the Statistical Package for Student version 17.0 (SPSS Inc., Chicago, IL) software p <0.05 was considered to be statistically significant.

Effect of B.nigra on the cell viability in PC-3 cells
B.nigra reduced the viability of PC-3 cells in a dose-dependent manner (50, 100, 200 and 400 µg) concentration. 50µg dose did not respond in cell growth inhibition whereas 100, 200 and 400 could effectively reduce growth significantly (p<0.05). However, it significantly decreased the viability of PC-3 cells in 24 h with IC 50 values of 200 and 400 µg/ml, respectively ( Figure 1). Hence, 400 µg concentrations was used for further analysis of protein expression of apoptosis signaling cascade.

Effect of B.nigra on Bcl-2 and p-Bcl-2 protein expression in PC-3 Cells
To examine the status of intracellular signaling molecules in the B.nigra-treated cells, protein expression analysis was performed. B.nigra treatment significantly decreased (p < 0.05) the antiapoptotic proteins such as Bcl-2 and p-Bcl-2 ( Figure 2).
Effect of B.nigra on p53, Case pase-3 and caspase-9 Protein Expression in PC-3 cells B.nigra treated PC-3 cells showed a significant increase (p < 0.05) in p53, casepase-3 & 9 protein expression stating that B.nigra has play significant role in the regulation of tumor suppressor protein activation ( Figure 3).

DISCUSSION
Apoptosis is a key process in cancer development. Evading apoptosis is one of the hallmarks of cancer. The apoptotic pathway plays a major role in prostate cancer as well. Prostate cancer treatment initially starts with androgen withdrawal, where many of the cancer cells die through apoptosis. But in some cases the cells derive resistance to this treatment and leads to metastatic prostate cancer. 20 Hence, in this study we aimed to manage this advanced stage of cancer by targeting apoptotic pathway.
As we were targeting advanced stage of cancer, hormone-independent cell line PC-3 was selected for the study.
Intrinsic pathway of apoptosis is one of the pathways activated by many cytotoxic drugs. Bcl-2 members family play a major role in the intrinsic pathway and this family contains 25 pro and antiapoptotic members which interact to maintain the balance between newly forming and old dying cells. 21 These proteins are classified into three subfamilies. Antiapoptotic subfamily contains the Bcl-2, Bcl-XL, Bcl-w, Mcl-1, Bfl1/A-1 and Bcl-B proteins which suppress apoptosis, some proapoptotic proteins such as Bax, Bak and Bok, other pro-apoptotic proteins such as Bim Bad and Bid.
The antiapoptotic proteins are over expressed in prostate cancer. These proteins heterodimerize with pro-apoptotic protein such as Bax/Bak, which will prevent the cell death.
Drugs have been used for prostate cancer targeting the antiapoptotic proteins. These drugs induce the activation of tumor suppressor protein p53 thereby increase the expression of pro-apoptotic protein and reducing the expression of antiapoptotic protein. 22 The same mechanism was observed in the B.nigra treated the PC-3 cells of the present study. There was an increase in the level of p53 and decrease in the antiapoptotic proteins level of Bcl-2 and p-Bcl-2. This clearly denotes that B.nigra promotes apoptosis by the intrinsic pathway.
This hypothesis is well determined through the analysis of caspase expression. Caspases serve as a primary mediator of apoptosis located in the cytosolic space. 23 In the present study, the treatment of B.nigra could lead to the release of cytochrome c from mitochondrial space which would have been combined with an adaptor molecule apoptosis protease activating factor 1 and also with an inactive initiator caspase, pro-caspase-9 within a multiprotein complex called the apoptosome. 24 This apoptosome would have caused a series of caspase activation which starts from 9 to 3, 6, 7 that ultimately would have caused apoptosis. Hence, it clearly depicted that B.nigra activates apoptosis by the intrinsic pathway. It is noteworthy to suggest the following hypothesis for the mechanism for apoptosis caused by B.nigra.  In present study, B.nigra treated PC-3 cells showed a significant decrease in anti apoptotic makers (Bcl-2 and p-Bcl-2) and increase in the p53, caspase-3/9 in PC-3 cells recorded in the present study showing that B.nigra has potenail cytotoxic activity. In view of these findings Rigano, et al. 18 reported the cytotoxicity of B.nigra against other two human cancer cells, hepatoma HepG2 cell line and breast cancer MCF-7 cell lines.

CONCLUSION
The current study proved that Bollata nigra strongly inhibits the growth of prostate cancer cells by interfering with the apoptotic pathway. It down-regulated the antiapoptotic proteins while up-regulated the pro-apoptotic proteins and thereby paved the way to cell death. The caspase-9 and caspase-3 activation proved that B.nigra acts through intrinsic apoptotic pathway. Thus, B.nigra potent chemotherapeutics compound for treating prostate cancer. Further experiments through animal model and clinical trial will lead B.nigra to be a potent drug in future.

CONFLICTS OF INTEREST
None declared.