Habb-e-Asgandh Suppresses Cell Proliferation and Induces Apoptosis through Mitochondria Dysfunction in Multiple Myeloma Cells (RPMI8226)

Objective: This study was conducted to assess the anti-neoplastic properties of Habb-e-Asgandh in multiple myeloma cells (RPMI8226). Methods: Multiple myeloma cells (RPMI8226) were cultured according to the ATCC’s instruction. The anti-proliferative effect of HeA was assessed by MTT assay and proliferating cellnuclear antigen (PCNA) activity. Cell cycle analysis, cellular apoptosis, and mitochondria membrane potential analysis was done by flow cytometry. Total antioxidants, migratory potential, angiogenesis and inflammatory biomarkers were also estimated after treatment of RPMI8226 with HeA. Results: LD30 and LD50 dose of HeA was 0.3mg/ml and 0.5mg/ml respectively determined by MTT assay and also confirmed by a reduced PCNA activity. Cell cycle analysis of RPMI8226 cells revealed that sub-G0/G1 phase increases upon treatment with HeA alone or in combination with lenalidomide. Annexin V-FITC/PI is used to detect early apoptosis, late apoptosis and necrotic cells and results showed that percentage of apoptotic cells increased in RPMI8226 cells after treatment with HeA. Also, HeA induces loss of mitochondria membrane potential (MMP) in MM cells in-vitro as measured by cationic JC1 dye staining. Upon treatment, the abnormal overexpression of oncogenic protein, AKT serine/threonine kinase has also been reduced. Furthermore, anti-oxidants level also increased while migratory potential, angiogenesis and inflammation decreased in multiple myeloma cell line upon treatment with HeA. Conclusion: Collectively, our results demonstrated that integrative therapy of habb-e-asgandh efficiently eliminates the need to use higher dose of lenalidomide for multiple myeloma treatment.


Introduction
Multiple Myeloma (MM), a disease of malignant plasma cells, which is 1% of all neoplastic diseases and takes second place in occurrence in haematological malignancies (10-15%) (Abramson, 2018). Several novel potential therapeutic agents such as immunomodulatory drugs, proteasome inhibitors, cell surface molecule specific antibodies and autologous stem-cell transplantation (ASCT) are used for the treatment of juvenile patients with multiple myeloma (Nishida and Yamada, 2019;Wudhikarn et al., 2020). Various invitro studies have been conducted for discovery of novel therapeutic agents for treatment of multiple myeloma such as tanshinone I which modulates telomerase activity and expression of shelterin complex thereby inducing apoptosis (Kumar et al., 2018). As explored in our previous study, anti-angiogenic and antitumor activity of cyclooxygenase-2 could be targeted as new therapeutic approach for treatment of multiple myeloma patients (Khan et al., 2013). Despite the numerous attempts to treat MM, current therapeutics still diseases such as arthritis, impotence, amnesia, anxiety, cancer, neurodegenerative and cardiovascular diseases (Mirjalili et al., 2009;Upadhyay et al., 2011;Baliga et al., 2013;Rasheed et al., 2013;Ojha et al., 2014). In a prostate cancer cell line, ashwagandha proves to downregulate various pro-inflammatory gene expression, including interleukins (IL-6, IL-1β, HSP 70,and STAT2) and upregulates p38/MAPK-kinase expression (Srinivasan et al., 2007;Nishikawa et al., 2015;Rah et al., 2015).

HeA Formulation
The HeA has been formulated as described in Table  1 using a soxhlet apparatus. The individual components of the formulation HeA were procured from the Aligarh city (Baradwari) in the month of August 2019 and were properly identified according to the botanical and Unani literature. These were also authenticated by the National Institute of Science Communication and Information Resources (NISCAIR), New Delhi with the voucher no. NISCAIR/ RHMD/ Consult/2020/3646-47-12 to 3646-47-19. The solubility of HeA has been tested in various solvents and 1X phosphate buffer saline (PBS) was found to have better solubility and hence, used for in vitro experiments.

GC-MS Characterisation of HeA
0.1g of HeA was dissolved in 1ml of DMSO followed by filtration using 0.22µ syringe filter and used for GCMS analysis. The phytocomponents were separated in zebron (Zb-5) column with column length 30 metres in GCMSQP2010, SHIMADZU instrument.

Cell Maintenance of RPMI 8226 cell line
The ethical approval for the cell line work was obtained from the Institutional Ethics Committee of AIIMS, New Delhi (IEC-539/02.12.2016).
RPMI 8226 cells were purchased from ATCC and cultured according to the manufacturer's instruction. The cells were also authenticated by STR typing in 2018 by DNA Forensics Laboratory, New Delhi, India. RPMI 8226 cells were maintained in complete RPMI1640 (Himedia, catlog no. AL162S) medium containing 10% Fetal Bovine Serum (Himedia, catalog no. RM9954) in an incubator at 37˚C with 5% CO2. When cell confluency reached 70% cells were incubated in complete medium for 24 h and then treated with different concentrations of HeA for 48 h. Mycoplasma testing (Abcam, catalog no. ab289834) was done in RPMI8226 cells and culture was found to be negative.

Measurement of cellular metabolic activity by MTT Assay
The cytotoxic effect of HeA was assessed by MTT assay. In brief, 1 x 10 4 cells were seeded in 96-well plate and incubated overnight at 37˚C with 5% CO2. Cells were then treated with different concentrations of HeA for different time points (24, 48, 72 h). After respective time period, 10µl of 5mg/ml Thiazoyl blue tetrazolium bromide (MTT) (catalog no. M5655, CAS No. 298-93-1, Sigma Aldrich) was added to each well including control and incubated at 37˚C with 5% CO 2 for 4 h. After 4h incubation, when formazon crystals (purple colour) was clearly visible, plate was centrifuged at 1500 rpm for 10 minutes. Then, supernatent was carefully removed and settled crystals were dissolved in DMSO (solubilisation solution) and plate was gently shaked for 20 minutes at room temperature. The absorbance was then measured at 570nm with reference wavelength of 630nm in a microtiter plate reader. LD30 and LD50 doses of drug were determined at which cells were 70% and 50% viable, respectively.

Detection of Cell Cycle Arrest
RPMI8226 Cells at 60-70% confluence were treated with LD30 and LD50 doses of HeA (HeA30, HeA50), LD30 and LD50 doses of Lenalidomide (standard drug) (L30, L50) and combinatorial doses of HeA30+L30, HeA30+L50, HeA50 + L30, HeA50 + L50 for 48 h. The cells from respective wells were then collected, fixed with 70% ethanol, washed and incubated with RNase A and propidium iodide (catalog no. P4170, CAS No. 25535-16-4, Sigma Aldrich) for 30 minutes at room temperature in dark. The cells were then analysed in flow cytometry. Kit with PI,Catalog No. 640914,Bio legend) Drug treated cells were resuspended in 100µl of Annexin V Binding buffer, 4µl Annexin-V-FITC dye and 8µl PI were added to cells, followed by incubation of 20 minutes and analysed by flow cytometry.

Detection of depolarization of mitochondrial membrane
RPMI8226 Cells were harvested 48 h post drug treatment and stained with 2µM of JC1 dye solution (catalog no. T4069, CAS no. 3520-43-2, Sigma Aldrich) and incubated for 30 minutes at 37˚C in dark. The stained cells were analysed by flow cytometry.
Cell Migratory Potential 1.5 X 10 5 drug treated RPMI8226 cells in 150µl in serum free media were added in the trans-well insert of 24-well migration plate. 500µl of complete culture media (with 10% FBS) was added to the lower well of migration plate, followed by an overnight incubation at 37˚C with 5% CO 2 . The percentage of migrated cells was calculated.

Oxygen Radical Absorbance Capacity (ORAC) Assay
Anti-oxidant potential of HeA was performed by ORAC Assay Kit (catalog no. ab233473, Abcam) and was performed as per the manufacturer's guidelines. To perform this assay, the ELISA plate reader was equilibrated followed with steps as given in the protocol. Concentration of the analyte is directly proportional to the intensity of final yellow coloured product. The optical density was then measured and plotted against the concentration to get a standard curve and a linear equation which can further be used for estimation of concentration in unknown sample.

Statistical Analysis
The data analysis was done using GraphPad Prism 6.0. All data results were presented as mean + SD. Different groups were compared using Student's t-test. Differences witha probability value of * p < 0.05 were considered statistically significant.

GC-MS Characterisation of HeA
The GC-MS chromatogram of HeA aqueous extract recorded a total of 60 peaks corresponding to bioactive compounds that were recognised by relating their peak retention time, peak area (%), height (%), and mass spectral fragmentation patterns to that of the known compounds described by National Institute of Standards and Technology (NIST) library. These compounds majorly include class of siloxanes, hydrolylamines, hetrocyclic organic compounds, cyclical aldehyde, acyclic olefins, sesquiterpene, amino acid carbamates, phthalates, aminosterol, niflumic acid, urocanic acid. GC-MS fingerprint chromatogram ( Figure 1S) along with list of bioactive phytoconstituents (Table 1S) is provided in to 37˚C. 25ul of samples (treated cell lysates) or Trolox standard was added to the respective wells, followed by addition of 150µl working fluorescein solution. The plate was kept at 37˚C incubator for 30 minutes. Then 25µl free radical initiator working solution was added and fluorescence kinetics was started and continued for 60 minutes. Then, standard curve was plotted between trolox concentration and Net AUC to determine the equation that was used to calculate the trolox equivalent of the sample.

Immunoblotting
After 48 h of drug treatment, cells were harvested and lysates were prepared in RIPA lysis buffer and protein concentration of the respective lysates was quantified using Bradford assay. The denatured proteins were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membrane was incubated with the respective primary and secondary antibodies. Chemiluminescence of the blots was observed in Azure Biosystems c280 and intensity of the bands was quantified using ImageJ software.

Enzyme Linked Immunosorbent Assay (ELISA)
A high sensitive sandwich ELISA kit was employed for the estimation of the level of Th1 (IL2), Th2 (IL-4, IL-10) and pro-inflammatory (IL-6) to investigate the anti-inflammatory role of HeA in cell culture medium supernatant. Standards and samples were added to the pre-coated micro-titre plate provided in the kits and  Habb-e-Asgandh regulates cellular metabolic activity To initiate the study of identifying anti-tumor potential of HeA, firstly, multiple myeloma (MM) cell line (RPMI8226) was treated with HEA and standard chemotherapeutic drug (Lenalidomide for MM) for 48 hours to determine sub-lethal dose (LD30) and lethal dose (LD50) of these drugs using MTT assay ( Figure 1 and Table 1). The standard drug, lenalidomide was not showing any cytotoxicity at any concentration, hence, as per the published literature, two doses of 5µM and 10µM were chosen for further experiments. All the experiments were performed in triplicates on these doses.

Morphological changes in myeloma cells (RPMI8226) upon treatment with Habb-e-Asgandh and standard chemotherapeutic drug
The phase contrast microscopy images in Figure 2 showed the combinatorial effect of drug treatment on cell toxicity and morphology. The cells appeared with visibly distorted morphologies and membrane blebs as shown in Figure 2 (A). The confocal microscopy of DAPI stained RPMI8226 cells also reveals morphological distortion and nuclear fragmentation as shown in Figure 2(B).
Then, we confirmed the cell cycle arrest in RPMI8226 Cells. HeA and combinatorial treatment increases the relative proportion of cells in sub G0/G1 phase. It also increases the proportion of cells in G0/G1 phase upon treatment with HeA50, HeA50 + L30 and HeA50 + L50, therefore indicating the cell cycle arrest at the G0/ G1 phase. Treatment with HeA alone or combinatorial treatment significantly reduced the proportion of cells in G2/M phase as shown in Figure 4.

HeA accelerates apoptosis in RPMI8226 cells
Followed by cell cycle analysis by PI staining, the mode of cytotoxicity of Habb-e-Asgandh was investigated, i.e., the cell death caused by drug treatment occurred by apoptosis or necrosis. For this, Annexin-V-FITC/PI assay was performed. In this assay, viable cells do not show any fluorescence, early apoptotic cells show only green fluorescence while late apoptotic cells show dual fluorescence. It has been observed that both early and late apoptotic cells increase upon treatment with Habb-e-Asgandh and standard drugs alone at dose dependent manner in RPMI 8226 cells, however, proportion of apoptotic cells further increase when both the drugs were given in combination as shown in Figure 5.

HeA hampers mitochondrial homeostasis in RPMI8226 cells
We assessed the relative mitochondrial membrane potential (MMP) using JC1 dye. HeA treatment alone or in combination with standard Lenalidomide drug increased the loss of MMP compared to control RPMI8226 cells as shown in Figure 6 (A, B, C).

HeA reduces the migratory potential of myeloma cells
An essential step towards the invasive potential of cells is cell migration. Results indicate that Habb-e-asgandh in combination with standard drugs substantially reduced the migratory ability of RPMI8226 cells (Figure 7). Therefore, by regulating the progress and migration of cancer cells,    integration of HeA with standard lenalidomide may acts as the anti-metastatic herbal drug.

ROS generation is induced by HeA in RPMI8226 cells
ORAC assay was performed to determine the anti-oxidant potential of Habb-e-Asgandh alone or in combination with standard drug in myeloma cells. Firstly, standard curve was plotted with the known concentration of anti-oxidant (Trolox) supplied in the kit to obtain a linear equation. This equation was then used to determine the anti-oxidant concentration in the unknown samples.
The results showed that the levels of trolox equivalent which is a measure of anti-oxidant in RPMI8226 cells treated with higher concentrations of Habb-e-asgandh were significantly higher. HeA induced significant ROS generation in RPMI8226 cells. These data indicates that one of the possible mode of anti-cancer activity of Habb-e-asgandh against RPMI8226 cell line is intracellular ROS generation (Figure 8).

HeA regulates expression of various proteins leading to activation of apoptosis pathway
To confirm the phosphorylation of signalling proteins affected by HeA treatment, we conducted western blot analysis of various signalling proteins. HeA dosedependently down regulated the expression of BCl2 protein which leads to activation of programmed cell death. Also, HeA treatment alone or in combination with standard drug supressed the expression of proteins like ERK, pERK, AKT, pAKT, STAT, pFAK (Figure 9).

HeA effects production of cytokines in RPMI8226 cells
Treatment of RPMI8226 cells withHabb-e-Asgandh alone or in combination with standard drugs affects inflammation. For this, the ELISA kits was used for estimation of the level of Th1 (IL2), Th2 (IL-4, IL-10) and pro-inflammatory (IL-6)and the equation obtained was then used to determine the concentration in the cell supernatent after treatment ( Figure 10). Inflammatory cytokines IL2, IL4 and IL6 were found to be decreased and anti-inflammatory cytokine i.e. IL-10 level was significantly increased.

Discussion
Multiple Myeloma (MM) has been known debilitating diseases from many centuries across the world and numerous chemotherapeutic agents are being used for their treatment.In spite of having surfeit of treatment regimen, relapse of disease after a short period of remission is of great concern. Hence, partial recovery along with toxic side effects of chemotherapeutic drugs in patients prompts to identify a potent drug with least toxicity. In Indian traditional medicine, herbal medicines are being used for the treatment of diseases including cancer (Patterson et al., 2007).
In this present study, translational application of polyherbal Unani formulation, Habb-e-Asgandh has been explored. GC-MS Characterization of HeA shows presence of various organic compounds, siloxanes, alkanes, long chain fatty acids, amino acid carbamates, flavanones, oxysterols, aromatic carboxylic acid, hydroxylcinnamic acid as its bioactive constituents which showed anti-tumor, pro-apoptotic, anti-inflammatory, anti-proliferative and anti-metastatic activities in previous research as listed in Table 2S (Harada et al., 2002;Lin et al., 2010;Yin et al., 2013;Mattarei et al., 2015). HeA decreased cell proliferation, PCNA expression and MMP in multiple myeloma cells (RPMI8226). Also, Oxygen Radical Absorbance Capacity (ORAC) has been found to be increased in RPMI8226 cells after treatment with HeA which shows increase in ROS production. Thus, HeA induced mitochondrial dysfunction and anti-proliferative effects on RPMI8226 cells by regulating signalling pathways. Therefore, in this study, we elucidated anticancer effects of HeA on RPMI8226 cells and their cellular mechanisms.
Ashwagandha has also been evaluated in oncology settings. In an open-label study, 100 patients with breast cancer were given a standardized extract (2 g every 8 hours) throughout chemotherapy. Researchers reported reduction in chemotherapy-induced fatigue (P < 0.001 with Piper Fatigue Scale; P < 0.003 with Schwartz Cancer Fatigue Scale) along with improvement in quality of life (P < 0.001) (Biswal et al., 2013). Larger trials are needed to validate these findings, establish an optimal dose, and determine long-term safety.In addition, microarray analysis revealed that Ashwagandha repressed proinflammatory gene expression, including interleukin-6 (IL-6), IL-1β, IL-8, heat shock protein 70, and STAT2, and induced p38/MAPK-kinase expression in a prostate cancer cell line (Aalinkeel et al., 2010). The root extracts were shown to exert cytotoxic effects in lung, colon, central nervous system, and breast cancer cell lines (Jayaprakasam et al., 2003). Active constituent of Ashwagandha demonstrated anticancer effects in leukemia cells (Malik et al., 2007)as well as in estrogen receptor-positive and estrogen receptor-negative breast cancer cells, via inducing apoptosis and decreasing tumor size .
Ashwagandha was proved to be selectively cytotoxic to cancer cells by ROS signalling (Widodo et al., 2010). Complementary to the prior study, in this research, we found that habb-e-asgandh (HeA) caused cytotoxicity of myeloma cells in vitro in dose dependent manner. Ashwagandha causes G2 and M phase cell cycle arrest in human breast cancer cells , similarly, upon cell cycle analysis, this has been observed that sub-G0/G1 phase increased upon treatment with HeAalone or in combination with standard drug, Lenalidomide, suggesting the cell death caused by the formulation and inducing cell cycle arrest at G2/M check point thereby increasing percentage of cells in G2 phase. The mode of cytotoxicity has been assessed by Annexin V-FITC/ PI assay which showed that percentage of apoptotic cells got increased in RPMI 8226 cells after treatment with habb-e-asgandh. In addition, mitochondrial membrane potential (MMP) determination by JC1 assay showed the loss of MMP after treatment. Furthermore, anti-oxidants level also increased while migratory potential, angiogenesis and inflammation decreased in myeloma cells upon treatment with HeA. In combination with standard chemotherapeutic drugs (lenalidomide), habb-e-asgandh further affected the morphology of RPMI8226 cell line, induces cell cycle arrest at G1/S check point leading to increase in cell number in G1 phase of cell cycle, persuading cellular apoptosis, increasing cytokine production and inhibiting cell migration in myeloma cells.
Conclusively, our study is the first to demonstrate the anticancer effects of Habb-e-Asgandh on RPMI8226 cells and to elucidate their cellular mechanisms and action on mitochondrial functionby activating signalling pathways via ROS generation and MMP depolarization. Also, we confirmedthat HeA suppressed proliferation via ROS generation in multiple myeloma cancer cells. HeA in combination with lenalidomide showed promising cytotoxicity in vitro. Importantly, HeA could be used as an adjuvant therapy with lower dose of lenalidomide as it showed favourable anti-proliferative effect, thereby, effectively eliminating the need to use higher dose of lenalidomide which could further reduce side effects related to standard therapy. Although our results arelimited to an in vitro evaluation, this study may provide valuable insights regarding theunderlying molecular mechanisms for future in vivo studies.

Supporting Information
GC-MS chromatogram of HeA ( Figure 1S) and list of bioactive phytoconstituents (Table 1S) along with their biological relevance (Table 2S) are available as supporting information.

Author Contribution Statement
AV, NG and AS designed the research study. AV performed the research and analyzed the data. NG provided assistance in few experiments. AV and AS drafted the manuscript. SN prepared the formulation, Habb-e-Asgandh as per unani guidelines. AS supervised the research, contributed essential reagents or tools, critically reviewed and edited the manuscript. All authors have read and approved the content of the manuscript.