Cytotoxic and apoptotic effects of heat killed Mycobacterium indicus pranii (MIP) on various human cancer cell lines

Mycobacterium indicus pranii (MIP) is a non-pathogenic mycobacterium, which has been tested on several cancer types like lung and bladder where tumour regression and complete recovery was observed. In discovering the potential cytotoxic elements, a preliminary test was carried out using four different fractions consisting of live bacteria, culture supernatant, heat killed bacteria and heat killed culture supernatant of MIP against two human cancer cells A549 and CaSki by 3-(4,5-dimethyl thiazol)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was investigated in MCF-7 and ORL-115 cancer cells by poly-(ADP-ribose) polymerase (PARP) and DNA fragmentation assays. Among four MIP fractions, only heat killed MIP fraction (HKB) showed significant cytotoxicity in various cancer cells with inhibitory concentration, IC50 in the range 5.6–35.0 μl/(1.0 × 106 MIP cells/ml), while cytotoxicity effects were not observed in the remaining fractions. HKB did not show cytotoxic effects on non-cancerous cells contrary to cancerous cells, suggesting its safe usage and ability to differentially recognize between these cells. Evaluation on PARP assay further suggested that cytotoxicity in cancer cells were potentially induced via caspase-mediated apoptosis. The cytotoxic and apoptotic effects of MIP HKB have indicated that this fraction can be a good candidate to further identify effective anti-cancer agents.

The use of bacteria in cancer treatment is a well-known approach which was championed by W. Coley and German physicians W. Busch and F. Fehleisen who reported recovery of neck and other cancers following an infection with Streptococcus pyogenes 1 . Following these discoveries, several other bacterial species have been found to elicit significant anti-tumour activity in both in vitro and in vivo systems such as Lactobacillus species on bladder cancer 2 and attenuated Salmonella species in murine tumour models 3 . Mycobacteria may be yet another promising species as it has shown a long successful history in treating cancer, for instance, the bacillus Calmette-Gue'rin (BCG) vaccine derived from Mycobacterium bovis was reported to be effective in treating human bladder cancer 4 .
Bacteria based anti-tumour therapy possess several advantages over chemical based drug. Firstly, some bacteria are able to selectively replicate and accumulate within tumour due to hypoxia environment and inhibits tumour growth. Next, motile bacteria are able to spread throughout the tumour and help in targeting systemic diseases. They can readily express multiple therapeutic transgenes such as cytokines and pro-drug converting enzymes to eradicate tumour mass 5 .
Mycobacterium indicus pranii (MIP) or conventionally known as Mycobacterium w (M.w) is a non-pathogenic, cultivable Mycobacterium species; which is now used widely as a vaccine for a number of diseases 6 . This vaccine works by boosting up the patient's immunity through the induction of CD4 + T helper 1 (Th-1) cells response to release cytokines IL-2, IL-12, IL-15 and IFN-γ in order to promote cell-mediated immunity, and has been reported to be safe for human use in the treatment of leprosy 6 , tuberculosis 7 , HIV infection 8 and lung cancer 9 diseases.
Apart from inducing the immune system, certain mycobacteria species were also reported to induce a direct cytotoxic effect on cancer cells. As reported by Saitoh and Morales, BCG and BCG components 4 could induce cancer cell apoptosis, while M. phlei or mycobacterial cell wall and DNA components possess certain anti-tumour activity 10 . These findings provides an insight on exploiting MIP and its cellular components as a potential anti-cancer agent against various human cancer cell lines.
As such to date, only certain types of cancers were reported to show cytotoxic effects upon MIP treatment in in vivo 11,12 . Therefore, there is a great interest to discover MIP cytotoxicity on various human cancer cell lines to broaden its utility. In this study, we evaluated in vitro cytotoxicity effect of four different MIP fractions consisting of live bacteria, culture supernatant, heat killed bacteria and heat killed culture supernatant against various human cancer type namely breast, cervical, oral, lung, bladder, liver and prostate.

Results
Cytotoxic screening for active MIP fractions. MIP was separated into four fractions: live bacteria (LB), culture supernatant (CS), heat killed bacteria (HKB) and heat killed culture supernatant (HKS). In identifying the active MIP fraction with cytotoxic effects, all four fractions were treated in two different cancer cell lines; cervical (CaSki) and lung (A549). The cell viability upon 24 hrs treatment was measured using MTT assay based on the mitochondrial activity in viable cells. In both cancer cells, only MIP HKB fraction showed cytotoxic effects where cell viability reduced to 24% in CaSki and 26% in A549, while the remaining fractions did not show any killing effects (Fig. 1). Thus, MIP HKB fraction was used here after to assess its cytotoxic consistency on various other cancer cell lines.

DNA fragmentation and PARP assay.
To further analyze the mode of cell death upon MIP HKB treatment, MCF-7 and ORL-115 cells were selected as model cell lines owing to it having an IC 50 value below the HaCat cell line threshold. The morphological changes in both cells shows MIP induced apoptotic cell death (data not shown). The PARP cleavage assay was carried out to validate the apoptosis mediated cell death in both cell lines. Results showed cleavage of the inhibitory fragment from the 116 kDa full length PARP into an 89 kDa fragment (Fig. 3). Cells were treated with PBS and MIP HKB, 12 μ l/(1.0 × 10 6 MIP cells/ml) for MCF-7 while 7.8 μ l/(1.0 × 10 6 MIP cells/ml) for ORL-115 in a time dependent manner at 6 and 12 hrs to observe the initiation and progression of apoptosis. The housekeeping gene, GAPDH was used as a protein normalization and loading control.
DNA fragmentation assay was carried out to confirm and observe the occurrence of late apoptosis in MCF-7 and ORL-115 cells at 6, 12 and 24 hrs. A 150 bp to 200 bp laddering of DNA at 12 hrs upon MIP exposure in MCF-7 indicates a strong hallmark of late apoptotic events (Fig. 4). Ladder formation was absent in both untreated and PBS treated cells, which showed that the appearance of apoptotic DNA fragments were due to the cytotoxic effect of MIP HKB treatment.

Discussion
In cancer treatment, MIP is used as an adjuvant to radiation therapy in patients with bladder cancer 12 and to chemotherapy plus radiotherapy in non-small cell lung cancers 10 . Four types of fractions can be obtained from MIP: LB, HKB, CS and HKS, with the most widely used fractions being the HKB fraction 13,14 and CS fraction 15 . While past studies have cited autoclaving for 20 mins at 15 lb/in 2 as the most common heat killing method, this method may denature important and biologically active proteins, which led us to heat-kill MIP at 60 °C, which was also found to be sufficient in killing MIP cultures. When all MIP fractions were cultivated in 7H10 agar, no growth was observed after a week of incubation, with the exception of LB fraction, thus confirming the complete killing of MIP at 60 °C. This method is recommended because even though MIP cultures were completely heat-killed, other intracellular and extracellular proteins/precursors potentially responsible for its cytotoxicity would likely remain intact. MIP HKB demonstrated therapeutic cytotoxicity against most of the tested human cancer cells, and was less potent towards non-cancerous human cells based on its high IC 50 value. The difference in MIP selectivity between non-cancerous cells and cancer cells may be due to differences in growth rate, which results from the presence of distinct cell surface receptors, differences in the uptake of certain drugs and the method used for assessment of toxicity 16  This study also identified that cancer cell death was induced via apoptosis in MCF-7 and ORL-115 cells as confirmed through PARP and DNA fragmentation assays. Apoptosis is a cell suicide mechanism to remove redundant, damaged, or infected cells through a group of caspases activation. These caspases are grouped into initiator (caspases-2, -8, -9, and -10) and effector (caspases-3, -6 and -7) caspases. Effector caspases are responsible for dismantling of necessary cell components, which results in morphological and biochemical changes that characterize apoptotic cell death as cytoskeletal rearrangement, cell membrane blebbing, nuclear condensation and  MTT cell viability assay. MTT assay was carried out to measure cytotoxic effects of MIP fractions on various cancer cell lines. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) is a substrate which is reduced by dehydrogenase enzymes present in the mitochondria of viable cells. In MTT assay, the intensity of the purple formazan product was measured and used to quantify viable cells in culture. A total of 100.0 μ L of cells were plated per well (1.0 × 10 4 cells/well), incubated overnight, and treated with MIP fractions at various concentrations then incubated for 24 hrs. 20 μ L of MTT reagent (5.0 mg/mL) was added to each well. The plate was left on a shaker for 10 mins and incubated in the dark at 37 °C. After 1 hr of incubation, the spent medium containing excess dye was aspirated and 200 μ L of DMSO added to dissolve the purple formazan precipitates. Results were obtained using micro-titer plate reader (Tecan Sunrise, Switzerland), to detect absorbance at a test wavelength of 570 nm, and a reference wavelength of 650 nm. From absorbance data obtained, a graph was plotted employing the following equation: Viability (%) = [100% -cytotoxicity (%)]; where cytotoxicity (%) = [(absorbance value of solvent -absorbance value of MIP fraction)/absorbance value of untreated cells] × 100%. IC 50 values for MIP HKB fraction were determined from the graph at 50% cell viability.
PARP cleavage assay. The occurrence of apoptosis was assessed based on the proteolytic cleavage of PARP by caspase-3. Briefly, 2.0 × 10 6 cells/mL were treated with MIP HKB (IC 50 ) and total proteins were extracted using the NE-PERW nuclear and cytoplasmic extraction kit according to manufacturer's protocol. Fractionation was done using SDS-PAGE and electro-transferred onto nitrocellulose membranes. All membranes were blocked with 5% w/v BSA, 1 × TBS, 0.1% Tween-20 at room temperature with gentle shaking for 90 mins, and incubated with primary antibodies: GAPDH (1:1000) and PARP (1:1000) overnight at 4 °C, followed by detection using HRP-conjugated secondary antibodies (Cell Signaling, USA), and Super Signal West Pico chemiluminescent substrate. Images were captured using the Fusion FX7 imaging system (Vilber Lourmat, France). Apoptosis was represented by cleavage of 116 kDa full length PARP into an 89 kDa product.
Scientific RepoRts | 6:19833 | DOI: 10.1038/srep19833 DNA fragmentation assay. Cells were treated with 1x PBS and MIP HKB at 6, 12 and 24 hrs before harvesting, and total DNA was extracted from both untreated and treated cells using the Suicide Track TM DNA Ladder isolation kit according to the manufacturer's protocol. MCF-7 cells treated with the apoptosis inducing agent, 1'-(S)-1'S-1'-acetoxychavicol acetate (ACA) served as a positive control. Extracted DNA was analyzed on a 1.5% (w/v) agarose gel electrophoresis and stained with ethidium bromide. Fragmentation of DNA was observed under UV illumination and visualized using a gel documentation system (Alpha Inotech, USA).

Conclusion
Currently Mycobacterium indicus pranii (MIP) has only been tested on lung and bladder cancers with tumour regression and complete recovery observed. Its effects on other cancer cell types have yet to be determined. The MIP HKB fraction was identified as the most potent cytotoxic fraction compare to LB, CS and HKS in terms of its low IC 50 values and induction of apoptotic cell death in breast and oral cancer cells. Therefore the cytotoxic and apoptotic effects of MIP HKB in these two human cancer cells indicate that it can be a good candidate for further pharmacological studies to identify effective biologically active anti-cancer agents. In summary, this study has proven that MIP HKB, killed at 60 °C can inhibit the growth of various human cancer cell lines through activation of apoptosis.