Resensitization of Akt Induced Docetaxel Resistance in Breast Cancer by ‘Iturin A’ a Lipopeptide Molecule from Marine Bacteria Bacillus megaterium

Development of the resistance is the major problem in cancer therapy. Docetaxel is a taxol alkaloid that is frequently used in metastatic breast cancer. However, resistance often limits the usefulness of this drug in many breast cancer patients. Manipulation of resistant cells to re-sensitize to the therapeutic effect of docetaxel is current strategy to overcome this problem. Here, we have introduced ‘Iturin A’ as a potent chemosensitizer in docetaxel resistant breast cancer cells. Combination of Iturin A and docetaxel treatment significantly hampered the proliferation of docetaxel resistant MDA-MB-231 and MDA-MB-468 breast cancer cells. Cell cycle analysis also showed massive amount of apoptotic population (Sub G0/G1) in combination therapy. A number of apoptotic and anti-apoptotic proteins were significantly altered in dual drug treated groups. Caspase 3 dependent cell death was observed in dual treatment. Molecular mechanism study showed that over-expression of Akt and its downstream signaling pathway was associated with docetaxel resistance. Iturin A significantly reduced Akt signaling pathway in resistant cells. This mechanistic action might be the reason behind the chemo-sensitization effect of Iturin A in docetaxel resistant breast cancer cells. In conclusion, Iturin A resensitized the resistant breast cancer cells to docetaxel therapy by inhibiting Akt activity.

Breast cancer is the most frequently disease diagnosed worldwide. Therapeutic strategy depends on expression of a number of receptors including estrogen receptor, progesterone receptor and human growth factor receptor 2. The clinical benefits of hormonal therapy, chemotherapy and immunotherapy are highly co-related with the expression of above receptors.
Docetaxel is a FDA approved semi-synthetic taxol alkaloid molecule. It is used as 1 st line chemotherapeutic agent in metastatic breast cancer therapy. It displays cell cycle arrest and apoptosis in cancer cells by stabilizing and preventing depolymerization of microtubules. However, development of docetaxel resistance is major clinical problem in different cancers including breast cancer patients 1 . Activation of a number of survival signaling pathways has been reported to promote resistant phenotype in cancer cells in response to docetaxel treatment. Other factors are activation of P-glycoprotein, inhibition of apoptotic mechanism, metabolic alteration, changes in microtubule structure and binding efficiency of docetaxel to the microtubules 2 . P-glycoprotein or multidrug resistant protein 1 (MDR 1) is the major contributing factor for chemotherapeutic insensitivity.
Akt signaling plays a critical role in cancer associated phenomena including tumorigenesis, proliferation, invasion, metastasis and angiogenesis. Activated Akt pathway is also responsible for development of resistance in cancer cells to various chemotherapeutics drugs [3][4][5][6] . Moreover, Akt has been found to have positive co-relation with P-glycoprotein that is involved in drug efflux and multidrug resistance in cancer cells 7 . In previous report, Akt was found to be up-regulated in docetaxel resistant cancer cells 8 . So, Akt inhibition can lead to development of new therapeutics strategy to overcome chemoresistance. Some earlier report showed that some specific Akt inhibitors can resensitize cancer cells to various chemotherapeutics agents 9,10 . In breast cancer model Akt is the critical mediator for insensitivity of chemotherapeutic agents. High level of Akt in breast cancer cells was co-related with enhanced resistance to chemotherapeutic agents and tumor with more Akt expression are susceptible to recurrence and relapse 11 .
Microbial lipopeptides represent a unique class of pharmacologically active molecules 12 . In pharmaceutical application, lipopeptides particularly daptomycin, caspofungin and viscosin are reported to have potential anti-bacterial, anti-fungal and anti-viral activities 13 . Recently, it was found that lipopeptides are also effective in multidrug resistant bacteria including methicillin-resistant Staphylococcus aureus and penicillin resistant Streptococcus pneumonia, gram negative bacteria and extremely drug resistant clinically isolates [13][14][15] . In our previous findings we isolated 'Iturin A' from marine bacteria Bacillus megaterium and tested its anti-tumor efficacy in various in vitro and in vivo models. It displayed anti-cancer efficacy by inhibiting Akt signaling pathway 16,17 . However, the effect of Iturin A on resistance cancer cells was not previously documented. In the current investigation, we systematically tested the potential efficacy of Iturin A in docetaxel resistant cancer cells. Our hypothesis was that due to Akt inhibitory effect, Iturin A could reverse the insensitivity of docetaxel resistant breast cancer cells.

Detection of early apoptosis in resistant breast cancer cells. Externalization of phosphatidylserine
was detected in treated resistant breast cancer cells. This biochemical phenomenon is associated with early apoptotic event. Our study showed that few annexin positive cells (green florescent) were present in Iturin A treated group. In docetaxel treated group less number of annexin positive cells was present. However in combined treated group, significantly high numbers of annexin positive cells were observed indicating elevated apoptotic induction due to resensitization effect of Iturin A ( Fig. 4A and B).  Inhibition of Akt signaling resensitized the resistant cells to docetaxel. Next we evaluated expression of P-Akt and its downstream signaling. Iturin A slightly inhibited P-Akt expression and docetaxel treatment   fails to inhibit P-Akt in resistant cells. Combination of Iturin A and docetaxel treatment significantly suppressed P-Akt expression. Expression level of total Akt protein was not significantly change among various groups. GSK3β is downstream protein of Akt signaling. We also tested phosphorylation status of GSK3β. Like P-Akt, similar kind of results was observed in P-GSK3β ( Fig. 6A and B).

Discussions
The menace of chemoresistance is the potential drawback during the clinical management of multiple cancers including breast cancer. During the early stage of treatment, chemotherapeutic drugs may be effective but gradually they display poor clinical response due to acquired resistance of cancer cells 18 . Some chemotherapeutic drugs are also less effective to cancer owing to inherent resistance of cancer cells 19 . Numerous molecular drivers in cancer cells were discovered previously expanding the knowledge domain of chemoresistance 20 . These findings enable the scientist to discover novel therapeutic approaches to overcome the chemoresistance. Docetaxel is currently drug of choice for treatment of locally advanced and metastatic breast cancer 21 . In addition, docetaxel is the drug of choices for triple negative breast cancer. So, in our study we selected highly invasive and triple negative breast cancer cells MDA-MB-231 and MDA-MB-468. Many cancer patients are unresponsive or less responsive to docetaxel therapy. Resensitization of resistant cancer cells with other agent to chemotherapeutic drug is established strategy. In this current investigation, we evaluated the effect of Iturin A in docetaxel resistant cancer cells.
Initially, we generated the resistant cells from sensitive cells (Fig. 1A). Characteristics cellular arrangement was observed in resistant cells by phase contrast (Fig. 1B and C) and rhodamine phalloidin staining (Fig. 1D and E). Large and multiple number of vesicles inside the resistant cells were observed. In addition to this, actin reorganization, increased cellular size and presence of multinucleations were also observed in docetaxel resistant cells. These altered morphological features were previously reported in resistant cancer cells including multiple vesicles, large and multi-neucleated cells 22,23 . Presence of large number of vesicles may prevent the cells from the lethal effects of cytotoxic drugs by employing different mechanisms 24,25 . Akt has prominent role in tumorigenesis, cell survival, radioresistance and chemoresistance 3,26 in multiple cancers. Expression of P-Akt was evaluated in sensitive and resistant cells. Our result showed that P-Akt expression was elevated in resistant MDA-MB-231R and MDA-MB-468R cells compare to sensitive cells ( Fig. 1F and G). Inhibition of Akt is accepted strategy to overcome resistance in cancer cell 27 . The sensitivity of Iturin A was tested in resistant cells by MTT assay (Fig. 2). The IC 50 value of docetaxel was determined in sensitive as well as resistant cells (Fig. 2). In resensitization assay,  (Fig. 2). Phase contrast microscopy was performed to check effect of Iturin A on MDA-MB-231R and MDA-MB-468R cells resensitization. Single treatment with Iturin A or docetaxel displayed very less apoptotic effect on MDA-MB-231R and MDA-MB-468R cells. However, the combination of these two agents showed significant level of apoptosis in resistant cells (Fig. 3A and B). Next, flow cytometry based cell cycle analysis was performed. Similar with previous result, individual treatment showed very less apoptotic population and combined treatment caused drastic apoptotic population in resistant cells (Fig. 3C and D). Externalization of phosphatidylserine is the critical marker for detection of early apoptotic events 28 . Here annexin/PI staining was performed to detect early apoptosis. Enormous numbers of early apoptotic cells (green florescent) were observed in combination treatment compare to individual treated group (Fig. 4A and B). Next we have performed colony forming capacity of resistant cells treated with Iturin A or docetaxel. Generally, colony formation assay determines cell reproductive death of cancer cells by anti-cancer agents measuring the effectiveness of chemotherapy 29 . Combined treatment displayed very low number of colonies reflecting potent cytotoxic action of this combination on reproductive ability of cancer cells (Fig. 4C and D). Apoptosis analysis at protein level was checked by western blot analysis. Bcl-2 protein is the critical component of tumor associated apoptosis event. Overexpression of Bcl-2 protein can promote proliferation tumorigenesis and inhibition of apoptosis in multiple cancers. In our findings, Iturin A and docetaxel potently suppress Bcl-2 expression in combination ( Fig. 5A and B). Another apoptosis related important protein is Bax. Bax down regulation play a critical role in cancer progression 30 . Apoptosis inducing chemotherapeutic drugs often up-regulate Bax expression in cancer cells 31 . In our study, significant Bax up-regulation was noticed in resensitized cells treated with Iturin A in combination with docetaxel (Fig. 5). Caspase activation is the necessary part of biological process of apoptosis 32 . Here caspase 3 activations The significance level was represented by *P < 0.05, **P < 0.01, ***P < 0.001.
were found prominent in combination therapy in resistant MDA-MB-231R and MDA-MB-468R cells (Fig. 5). Akt signaling pathway has been reported to promote cell survival, inhibition of apoptosis, resistance to multiple chemotherapeutic drugs like cisplatin, carboplatin, doxorubicin, 5-flurouracil, cyclophosphamide, paclitaxel and docetaxel 4,33-38 . Inhibition of Akt signaling can resensitize the cancer cells. Our study showed that inhibition of Akt by Iturin A treatment resensitized the resistant cells to docetaxel treatment. Expression of phospho Akt was reduced in Iturin A treated group. Docetaxel treatment was unable to inhibit P-Akt in MDA-MB-231R and MDA-MB-468R cells. However, drastic change of P-Akt expression was noticed in Iturin A along with docetaxel treated groups (Fig. 6). The downstream protein GSK3β is major Akt substrate. Phosphorylation state of GSK3β was also drastically downregulated in resensitized cells treated with Iturin A and docetaxel combination (Fig. 6).
Our findings offered a novel approach to overcome chemoresistance that is a major clinical problem in chemotherapy. Docetaxel treatment is the critical part of standard chemotherapeutic regimen of breast cancer therapy. However, insensitivity or resistance to docetaxel therapy is the challenging drawback for successful cancer management. Akt is frequently over-expressed in resistant cell population. In our study, Iturin A inhibited Akt and restored the sensitivity of resistant MDA-MB-231R and MDA-MB-468R breast cancer cells to docetaxel therapy (Fig. 7).  MTT assay. Anti-proliferative effect was determined by colorimetric based MTT assay according to earlier reported method 40 . In brief, MDA-MB-231, MDA-MB-468, MDA-MB-231R and MDA-MB-468R cells were seeded in 96 well plates. After overnight incubation, cells were treated with different concentration of docetaxel for 48 h. After treatment, medium was discarded and MTT solution (1 mg/ml in incomplete DMEM medium) was added and cells were incubated for 5 h. After incubation MTT solution was discarded and DMSO was added in each wells. MDA-MB-231, MDA-MB-468, MDA-MB-231R and MDA-MB-468R cells were seeded in 30 mm Petri dish. The morphology of sensitive as well as resistant cells was captured by phase contrast microscopy 41 . In another set of experiment, MDA-MB-231R and MDA-MB-468R cells were seeded in 30 mm Petri dish. After 70% confluence, cells were treated with Iturin A and/or docetaxel for 48 h. The dose of Iturin A was 4 µM and 5.6 µM Iturin A for MDA-MB-231R and MDA-MB-468R respectively. The dose of docetaxel was 50 nM for both resistant cell lines. After treatment, phase contrast images of treated resistant cells were captured by microscope. In next experiments, resistant cells were treated with above mentioned Iturin A and docetaxel.

Phase contrast microscopy.
Actin reorganization. Actin reorganization of resistant cells was visualized by rhodamine phalloidin/DAPI staining according to previously reported method 41 . Briefly, MDA-MB-231R and MDA-MB-468R cells were seeded on cover slips. After 70% confluence, cells were treated with Iturin A and/or docetaxel for 48 h. After treatment, cells were washed, fixed and stained with rhodamine phalloidin. Florescent images of treated resistant cells were captured by Zeiss Observer Z1 microscope (Carl Zeiss, Germany) at 20 × magnifications.
Cell cycle analysis. Quantitative apoptotic effect was determined by DNA content based flow cytometric analysis according to earlier reported method 42 . MDA-MB-231R and MDA-MB-468R cells were seeded in 60 mm Petri dish. Cells were treated with Iturin A and/or docetaxel for 48 h in incomplete medium. After treatment, cells were harvested by trypsin treatment. Collected cells were fixed in 70% chilled ethanol for overnight. Then, cells were stained with propidium iodide mixed with RNAse. Cells were kept at 37 °C for 30 minutes. Samples were subjected to flow cytometric analysis.

Annexin/PI staining. Externalization of phosphatidylserin (Marker of early apoptosis) was checked by
Annexin-FITC/PI staining in treated cells according to earlier reported method 43 . Briefly, MDA-MB-231R and MDA-MB-468R cells were seeded in 30 mm Petri dish. Cells were then treated with Iturin A and/or docetaxel for 48 h. After treatment, cells were harvested and cell suspension was stained using Annexin V-FITC apoptosis detection kit (sigma) according to manufacturer protocol. Cells were then placed on glass slides and florescent images were captured by Zeiss Observer Z1 microscope (Carl Zeiss, Germany) at 20 × magnifications.
Colony formation assay. Cellular reproductive growth of resistance cells were determined by colony formation assay as earlier reported method 29 . Resistant cells were initially treated with Iturin A and/or docetaxel for 24 h. Cells were harvested and 1000 cells were seeded in six well plates. After 7 days, number of colony was counted in each group. Each colony contains greater than 50 cells. After 70% confluence cells were collected, washed with PBS and lysed with NP-40 lysis buffer. After lysis cell debris was separated by centrifuge and clear supernatants was collected in fresh tube. Concentration of protein was estimated by commercially available protein estimation kit. Samples were prepared by adding 6x dye and boiling. Equal amount of protein was loaded in each wells and subjected to SDS PAGE gel electrophoresis. Protein was transferred to nitrocellulose membrane. Detection of protein expression was evaluated after using primary and secondary antibodies. In another experimental setup, resistant cells were treated with Iturin A and/ or docetaxel. Western blot analysis was performed to evaluate a number of protein expressions.

Statistical analysis.
All experiments were performed at least three times. Experimental values were presented as mean ± S.D. Graphpad Prism software was used to draw bar graphs. P value was calculated using student's t test. P value < 0.05 was considered statistically significant. P value was calculated in various experiments to compare significance among the various groups.