5. DISCUSSION Tremendous application potential exists in the field

5.     DISCUSSION

Tremendous application potential exists in the field
of drug discovery from microbial sources. A number of novel secondary
metabolites and pigmented compounds with wide range of bioactivities against
several diseases have already been identified from microorgansims. Apart from
being colorants, microbial pigments have deeply penetrated into food, pharmaceutical
and cosmetic industries. They possess biological properties such as anticancer,
antimicrobial, antiproliferative and antioxidant activities (Rao et al., 2017). There are various fungal
and bacterial species isolated from soil, water and marine sources that produce
a wide range of pigments like, prodigiosin, carotenoids, melanine, pyocyanine
and violacein having various applications (Malik et al., 2012). In order to find a natural, alternative therapeutic
compound to treat cancer, the present work was aimed at evaluating   the
cytotoxic potential of bacterial pigments. In this pursuit, we isolated
pigmented bacteria from soil sources in Bangalore and were successful in collecting
five pigmented colonies. Two yellow coloured bacteria among the five have
demonstrated promising cytotoxicity to the cancer cell lines HeLa, HepG2 and
Jurkat. These two were identified as Pseudomonas
stutzeri JGI 52 and Micrococcus
terreus 19 by molecular methods.

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The pigments from these bacterial isolates were
extracted and their anticancer effects were analyzed on in-vitro cancer cell lines, HeLa, HepG2 and Jurkat. As the pigment
extracts from the current isolates have demonstrated promising anticancer
potential, they were partially purified using thin layer chromatography
technique to identify the compound which exhibited cytotoxicity. The yellow
pigment extract from P. stutzeri gave
a distinct visible yellow band with an Rf value of 0.73, which was named as
PY3. The yellow pigment extract from M. terreus
also had resulted in a visible yellow band with an Rf value of 0.64 and this band was named as MY3.
As these two yellow fractions, PY3 and MY3, demonstrated anti- proliferative
effects on all the in- vitro cancer
cell lines, they were chosen for further studies. To analyze the safety aspects
on humans, these fractions were checked on normal human peripheral lymphocytes
and also on a non cancerous cell line, CHO. 

The yellow
pigment PY3 excerted cytotoxic and antiproliferative effects on the cervical
cancer cell line HeLa, liver cancer cell line HepG2 and leukemia cell line
Jurkat. There
was a dose and time dependent growth inhibitory effect of PY3 on all the cancer
cells. PY3 markedly decreased the viability (less than 50%) of the cancer cells
analyzed, with an IC50 value of < 10 µg/ml (7.6 µg/ml on HeLa, 8.2 µg/ml on HepG2 and 9.07 µg/ml on Jurkat). At the same time, it had no or least toxicity to lymphocytes and CHO cells at the tested concentrations. This property is of greater significance towards the development of a therapeutic agent, which is safe for normal cells, safety being a major concern in the field of cancer chemotherapy. By chemical screening method, PY3 was found to be carotenoid. This is the first report of a yellow carotenoid pigment from P. stutzeri with anticancer activity. According to an earlier report, Pseudomonas stutzeri HMGM-7 produced a melanin pigment which was reported to possess antioxidant and cytotoxic activities. In-vitro cytotoxicity of melanin pigment was found against two human cancer cell lines A549 (human epithelial carcinoma) and HeLa (human epithelial cervical cancer) and the percentage viability of A549 reported was around 75% at 500 µg/ml of concentration.  Percentage cell viability of HeLa cells was 85% at 500 µg/ml of concentration (Ganesh et al., 2013). As compared to these reports, the carotenoid pigment PY3 in the current study had higher cytotoxicity against HeLa and HepG2 cells with 30% viability for HeLa and 35% viability for HepG2 cells when treated at 20 µg/ml concentration. In another report, marine Pseudomonas aeruginosa GS-33 produced a pigment PCA (Phenazine-1-carboxylic acid), which was having anticancer activity against human skin melanoma cell line SK-MEL-2. According to the report, IC50 value of the pigment against SK-MEL-2 cell line was < 10 µg/ml (Patil et al., 2016). In another study, a blue-green pigment pyocyanin from Pseudomonas aeruginosa was reported to have antiproliferative properties against HepG2 cells and responsible for ciliary dismotility and neutrophil apoptosis. At the concentration of 10 µg/ml of pyocyanin treatment, there was approximately 67% reduction in the cell numbers (Zhao et al., 2014). When compared with this value, by the treatment of the pigment PY3 from P. stutzeri, the percentage reduction in the cell number of HeLa and HepG2 cells were 70% and 65 % respectively. This very clearly suggests that the pigment PY3 in the current study has higher cytotoxicity than the pigment pyocyanin. The yellow pigment MY3, extracted from M. terreus, had significant growth inhibitory effects on HeLa, HepG2 and Jurkat cells at very low concentrations, with an IC50 value < 15 µg/ml  on all the cancer cell lines (10.24 µg/ml on HeLa, 12.4 µg/ml on HepG2 and 11.3 µg/ml on Jurkat). With respect to the normal cells (CHO and lymphocytes), MY3 treatment resulted in no cytotoxicity at the tested concentrations and durations. On exposure to increasing concentration of these cytotoxic pigments from low to high (0.25 to 20 µg/ ml) and with increase in the duration of exposure (24 to 72 h), percentage viability of the cancer cells decreased. This shows that percentage of the viable cells was inversely proportional to increasing concentrations of the cytotoxic compound and increase in exposure time. This property well suits the requirements towards the development of any anticancer drug molecule. This is the first report of a pigment from M. terreus having cytotoxic properties against cancer cell lines, through other species of Micrococcus have been reported as to have anticancer activities. There was a report about the ethyl acetate extract of M. luteus with a metabolite named as Pyrrolo 1, 2-a pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl), possessing anticancer activity against colorectal adenocarcinoma cancer cell line (HCT 15) and human uterine cancer cell line (MES-SA) (Mithun et al., 2012). Further, there is another report of a yellow pigment from M. luteus having cytotoxic effects against breast cancer cell line (MCF-7) (Ushasri et al., 2015). Rostami et al., (2016) reported about a carotenoid pigment produced from Micrococcus roseus (PTCC 1411) showing antioxidant and anticancer properties. Further proof for the cytotoxic and anticancer potential of the pigments came from the results of trypan blue assay, which is an accurate method for the determination of number of viable and dead cells. Treatment with PY3 for 24 h resulted in reducing the total cell count of HeLa cells to less than 50%, as compared to the control (from 6.0×105 to 2.5×105 cells/ml). HeLa cell count after 24 h of exposure to MY3 also dropped from 6.0×105 to 2.8×105 cells/ml. Similar effect was seen when HepG2 cells were treated with PY3 and MY3. The results from the current study clearly indicate that the pigments PY3 and MY3, do indeed inhibit the proliferation of the treated cells, apart from merely killing them by direct cytotoxicity. Sensitivity of the cancer cell line to the anticancer compound is an essential parameter to be looked upon, while choosing chemotherapeutic agents. The cancer cell lines HeLa and HepG2 were sensitive to PY3 and MY3, which had direct cytotoxicity and were able to induce apoptosis in the treated cells as indicated by morphological observation under the inverted microscope and fluorescence microscopical observations after AO/EB staining. The results demonstrated a drastic reduction in cell numbers of both HeLa and HepG2, along with apoptosis of the treated cells evidenced by the presence of apoptotic bodies and cells fluorescing bright orange as opposed to the untreated control cells which were viable and fluorescing green. One of the major goals in cancer therapy involving external agents is inducing cell death via apoptosis and thereby inhibit the growth of the cancer cells. DNA fragmentation being the hallmark of apoptosis, can be used to demonstrate induction of apoptosis in the treated cancer cells. The pigments in the current study, PY3 and MY3, were able to induce apoptosis in the treated HeLa and HepG2 cells as there was a smear of DNA when the DNA extracted from the treated cells were electrophoresed in an agarose gel. The DNA pattern on the gel gave a clear evidence for cells undergoing apoptosis. Montaner et al., (2000) have found a characteristic ladder pattern in the DNA of all the haematopoietic cancer cell line when treated with 10 µg/ml of CS-2170 (Serratia marcescens 2170). Whereas, in the present work both the pigments PY3 and MY3 treatment resulted in a smear pattern in the DNA of the HeLa and HepG2 cells. Furthermore, the onset of apoptosis involves the activation of caspase 9 in cells. Caspases are cystine aspartate proteases that get activated when a cell is damaged, which cleave their target proteins like the cytoskeletal elements, histones and others. However, they remain in an inactive state under normal conditions and activated only by an appropriate stimulus. Activation of caspase -9 is considered as an important biomarker of cells undergoing apoptosis, as they are the crucial mediators of apoptosis involving the activation of death proteases. This should be one of the important traits to be considered while searching for novel anticancer drugs. Hence, when caspase 9 activity was analysed on HeLa and HepG2 cells treated with PY3, a 3 fold and 2 fold increase in the percentage caspase activity respectively was observed when compared with that of the untreated cells. When they were treated with MY3, a 2.5 fold and a 3.7 fold increase in caspase activity of HeLa and HepG2 cells was observed respectively. In one study conducted by Deorukhkar et al (2008), N-alkylated prodigiosin analogue (MAMPDM), which is a pigmented compound, induced apoptosis in S-180 and EL-4 tumour cell lines. By assessing caspase- 3 activity he found 8-9 fold increase in the fluorescence intensity in the EL-4 cells treated with MAMPDM due to cleavage of substrate as compared to untreated cells. Many of the naturally existing anticancer compounds bring down the cell proliferation by direct cytotoxicity along with inducing apoptosis in the treated cells. This cytotoxicity can be well demonstrated by lactate dehydrogenase activity assay. This cytosolic enzyme is released to the culture media when the cell membrane is damaged. Here in the current study, the pigments PY3 and MY3 were able to cause damage to the plasma membrane of the treated HeLa and HepG2 cells. There was an increase in the LDH activity in the HeLa and HepG2 cells when treated with the pigment PY3 and the percentage cytotoxicity was 62.8 and 55.6% respectively. The pigment MY3 had relatively higher cytotoxicity for HepG2 cells which was 66.3%, and for HeLa cells it was 63.2%. It appears that both PY3 and MY3 have direct cytotoxic effects too along with their ability to induce apoptosis in the HeLa and HepG2 cells, when the results of all the assays were analysed to find out the mechanism by which these two compounds were bringing down the viability percentage of the treated cells. As the pigments from P. stutzeri and M. terreus were found to have anticancer significance, an attempt was made to optimize the culture conditions and media composition for maximising their yield. Media composition plays a major role in enhancing the production of metabolites, pigments or enzymes of commercial significance. The important culture conditions such as temperature, pH and incubation period along with incorporation of different media components like carbon, nitrogen and metal ions, were chosen to see their impact on pigment production. It was found that maximum pigment production from P. stutzeri was at pH 7.5 and that from the M. terreus was at pH 7.0. This is not surprising, as in some earlier reports also it was stated that pH 7.0 gave highest undecylprodigiosin accumulation in Streptomyces sp. JS520 (Stankovic et al., 2012). When coming to the incubation temperature, it was seen that maximum pigment production from both P. stutzeri and M. terreus was at 37°C. Stankovic et al. (2012), reported the highest biomass and pigment yields at 30°C by Streptomyces sp. JS520. In another study, Antony et al., (2011), reported that Serretia marcescens SU-10 produced more prodigiosin at 28°C and the rate was reduced as the temperature was increased. Williams and Quadri (1980) also found that prodigiosin production was best at 27°C and when temperature was shifted to a higher range at 38°C, there was no prodigiosin production. Unlike the red pigment prodigiosin which is produced at lower temperatures (27-30°C), the yellow pigments PY3 and MY3 of the current study were produced maximally at 37°C, which is also the most ideal temperature for the growth of majority of the bacteria. When the effect of incubation period on pigment yield was studied, a 72 h period was found ideal for maximum pigment production from P. stutzeri as well as M. terreus.  Incorporation of media with different carbon, nitrogen and metal ion salts greatly influenced the pigment production. P. stutzeri produced maximum pigment in a media supplemented with lactose and there was a 1.5 fold increase in pigment production. Sucrose was responsible for enhanced biomass and pigment production from M. terreus and resulted in a 1.4 fold increase. In some earlier studies, Subhasree et al., (2011) reported that media supplemented with fructose as a carbon source highly influenced the production of red and yellow pigments from Monascus purpureus. Chang et al., (2000) reported the maximum production of prodigiosin in a media supplemented with dextrose. Sundaramoorthy et al., (2009) found that Serratia marcescens produces more prodigiosin in a medium supplemented with maltose. Oller (2005) reported the repressive effect of glucose and sorbitol on prodigiosin synthesis. These reports suggest that different pigments have different biosynthetic pathways for their production from different microbes and that is why the great variation in their requirements of carbon sources for pigment biosynthesis. Among the nitrogen sources tested, beef extract induced maximum pigment production from P. stutzeri. Peptone was the second best source which supported with significant pigment production. When pigment production from M. terreus was analysed, media supplemented with yeast extract gave best results with a 2 fold increase in pigment yield. When we compare these results with prior studies, as per one study, Palanichamy et al., (2011) reported that sodium caseinate and peptone were suitable substrates for pigment production from Streptomyces sp. and sodium casinate provided highest biomass yield (0.058 gm/ml). Ammonium chloride was reported as a better inorganic nitrogen source for pigment production from Monascus purpureus (Chen and Johns, 1993; Juzlova et al., 1996). But in the current study, casein and ammonium chloride both were having repressive effects on pigment production by M. terreus. Thus the differences in nitrogen requirement again are indicative of differential biosynthetic pathways of the pigments. Among the different metal ions tested, highest pigment production from P. stutzeri was achieved when the media was supplemented with magnesium as 1% MgSO4. But, in the case of M. terreus, none of the metal ions augmented pigment production. In the present study conventional method was successfully adopted for initial screening of the culture parameters and their impact on pigment production but the main disadvantage of this method is that we could optimize only one parameter at a time. It is a time consuming and a laborious process which requires more number of experiments. Hence, a shorter and more reliable method, the statistical optimization through response surface methodology (RSM), was adopted to evaluate the interactive effects of variables and to select the optimum media components for maximizing the yield of the cytotoxic pigments. RSM usually involves an experimental design such as Central Composite Design (CCD) that provides a systematic plan for designing experiments, evaluating effect of target factors and standardizing optimum conditions of different variables for obtaining desired and improved response (Rani et al., 2011). This process consists of a second order polynomial equation that is used to describe the test variables and the combined effect of all the test variables in the response (Myers et al., 1995). Through RSM, the optimum culture condition for obtaining the maximum yield of the pigment from P. stutzeri was determined and the corresponding values of significant variables were found as 0.52% lactose, 1.01% beef extract, 1.003% MgSO4 and a pH of 7.55. The maximum pigment yield predicted was 2.89 mg/ml through this model, which was 2.5 fold higher than the yield through unoptimized conditions. In case of M. terreus, the optimum culture conditions were 1.068% sucrose, 1.11% yeast extract, 0.01% FeCl3 and a pH of 7.46 towards a pigment yield of 3.26 mg/ml, which was about 3 fold higher than that of the control conditions. At optimum physical and culture parameters, in just 30 runs, it was possible to get a significant increase in pigment yield, which indicates that this statistical design model offers a valid and efficient approach for enhancing the production of these useful compounds. As the two pigments PY3 and MY3, were of therapeutic significance, it was found necessary to chemically characterize them. The chemical screening of the pigment PY3 indicated the presence of carotenoids which was confirmed by IR spectroscopy. For further characterization, when it was subjected to LC-MS analysis, 6 fractions eluted at different retention times (RT) through liquid chromatography. The major eluent with 100% abundance was at 15.39 minutes. The mass spectrometric analysis of this major peak resulted in several fragment ions, out of which one fragment had an m/z value of 634.45 which corresponds to the compound fucoxanthinol with a molecular weight 616.42. Fucoxanthinol is a carotenoid and important studies in support of its contribution in cancer research already exist. Fucoxanthinol is a carotenoid anticancer compound isolated from brown sea-weeds (Kumar et al., 2013). As per their report, this is a potent cytotoxic agent against various cancer cell lines such as HeLa, HepG2, HL-60, HT-29,PC-3, PEL, Caco-2 and DLD-1. In an earlier study, Liu et al., (2009) reported the antiproliferative effect of fucoxanthinol against human hepatoma and murine embriyonic liver cells. In another study, Hosokawa et al., (2004), reported the anticancer effect of fucoxanthinol on colon cancer cell lines (Caco-2, DLD-1 and HT-29) and a significant decrease in cell viability as compared with other carotenoids like astaxanthin and ?- carotene. Ganeshan et al., (2011) reported an antiproliferative and apoptotic effect of fucoxanthin and siphonaxanthin, the marine carotenoids, on leukemia cells (HL-60). Though there are reports like this about fucoxanthinol from marine seaweeds, our study is the first one to report about this carotenoid from a terrestrial bacterial isolate, i.e, P. stutzeri JGI 52 with significant cytotoxicity to cancer cells. When the pigment MY3 from M. terreus was checked biochemically, the presence of terpenoids and steroids were indicated, which was further confirmed by IR spectroscopy. During further characterization by LC-MS analysis, liquid chromatography resulted in 8 fractions eluting at different retention times (RT). Mass spectrometric analysis of one of the eluents with RT 17.203 minutes, resulted in a fragment ion with a mass to charge ratio (m/z) of 337.2 which corresponds to the molecular weight of spiromustine. According to a study by Shoemaker et al., (1983), spiromustine demonstrated antitumour activity against a variety of tumours such as implanted B16 melanoma, implanted ependymoblastoma and colon tumours. The mass spectral data of another fraction eluted at RT 19.4 minutes, resulted in a fragment ion with an m/z value of 406.32, that corresponds to the compound bactobolin with a molecular weight of 383.33. In an earlier study, Kondo et al., (1979), reported antitumour activity of bactobolin on mouse leukemia cells, where bactobolin was found in the culture broth of Pseudomonas BMG13-A7. The presence of these two compounds i.e, spiromustine and bactobolin in the pigment fraction MY3, might be responsible for its currently observed anticancer activity. Though these two compounds were reported earlier from other sources, this is first report of their presence from Micrococcus terreus JGI 19. The current study demonstrated that the pigment fractions PY3 and MY3 from P. stutzeri and M. terreus respectively, have strong cytotoxic and antiproliferative properties against the cancer cell lines HeLa, HepG2 and Jurkat at very low concentrations and additionally are non toxic to normal human peripheral lymphocytes and CHO cell lines even at higher concentrations, which make them ideal candidates for future in-vivo and drug developmental studies. However, further characterization studies are required to identify the anticancer lead molecule responsible for their cytotoxicity.