Advances in Biology, Biotechnology and Genetics
Volume 3 | Issue 2 | Pages 01-08
Cytotoxic Activity of (2S, 5R, 6R)-2-Hydroxy- 3,5,6-Trimethyloctan-4-One Extracted from Marine costal soil Derived Streptomyces sp. VITDDK3
Krishnan Kannabiran *1 and Lakshmipathy Deepika 1
1School of Biosciences and Technology, VIT University, Vellore- 632014, Tamil Nadu, India.
The aim of the present study was to screen the cytotoxic activity of the extracted novel compound (2S,5R,6R)- 2-hydroxy- 3,5,6- trimethyloctan-4-one (HTMO) from marine Streptomyces sp. VITDDK3. The compound was extracted by bioactive guided extraction method, purified and identified by spectroscopic studies and named as HTMO with the molecular formula C11H22O2. The cytotoxic activity was studied against selected cancer cell lines. The cancer cell line HT-29 was sensitive to HTMO (5263 µM) with the IC50 value of 336µM, followed by HepG2 cells (IC50: 672 µM), MCF7 cells (IC50: 672 µM) and HEp-2 cells (IC50: 2688 µM). The compound HTMO (5263 µM) showed cytotoxic activity with the IC50 value of 2688 µM against VERO cells. Since the lead compound HTMO has showed cytotoxic activity against HT-29 cancer cells under in vitro conditions, it suggest that HTMO can be studied further to be used as cytotoxic agent against cancer cell lines
Keywords: Streptomyces sp. VITDDK3, (2S,5R,6R)- 2-hydroxy- 3,5,6- trimethyloctan-4-one, Cancer cells, Cytotoxicity
Among the diverse marine microbial communities, actinobacteria have occupied a prominent and significant position as potential producers of structurally complex and unique metabolites. Streptomycesare a prolific source of secondary metabolites yielded many antibiotics; more than 80% of antibiotics available in the market are from Streptomyces, including, streptomycin, neomycin, tetracycline and chloramphenicol (De Lima Procópioa et al., 2012). Several reports are available on the anticancer activity of compounds isolated from marine Streptomyces. Streptomyces produces clinically useful antitumor drugs such as anthracyclines (aclarubicin, daunomycin and doxorubicin), peptides (bleomycin and actinomycin D), aureolic acids (mithramycin), enediynes (neocarzinostatin), antimetabolites (pentostatin), carzinophilin, mitomycins and others (Newman and Cragg, 2007; Olano et al., 2009). The high toxicity usually associated with cancer chemotherapy drugs and their undesirable side effects increase the demand for novel antitumor drugs active against untreatable tumors, with fewer side effects and/or with greater therapeutic efficiency are very much required for the effective control and management of cancer (Demain and Sánchez, 2009). Actinomycetes have evolved with greatest genomic and metabolic diversity and hence efforts have been directed towards exploring marine Streptomyces as a source for the discovery of novel secondary metabolites (Udwary et al., 2007).
Streptomyces strain 23-2B isolated from the marine shellfish Donax trunculus anatinus collected at the Mediterranean Sea has been reported to possess high antitumour activity against Ehrlich’s ascites carcinoma (El-Shatoury et al., 2009). It showed selectivity towards solid tumours and high cytotoxicity to human carcinoma of liver (HEPG2), cervix (HELA) and breast (MCF7). Marine Streptomyces griseorubens isolated from marine sediment of China exhibited anti-tumour activity (Ye et al, 2009). The search for novel drugs is still a priority goal for cancer therapy, due to the rapid development of resistance to multiple chemotherapeutic drugs. Hence a study was planned to explore the cytotoxic property of HTMO against cancer cell lines.
Materials and Methods
Sampling site and collection of costal soil samples
The marine soil samples were collected from 11 different locations of Ennore saltpan at a distance of 1 km, (Lat. 13°.14’ N and Long. 80°.22’ E) located about 24 km north of Chennai, Tamil Nadu, India. Soil samples (~500 gm) were aseptically collected about 5-7 inches deep from the surface of the seashore using sterile spatula in sterile polyethylene bags along with sea water and transported to the laboratory under aseptic conditions. The collected samples were maintained at 4° C for further analysis.
Isolation of actinomycetes
Starch caesin agar medium (SCA) was used for the isolation of actinobacteria from soil sample. A tenfold serial dilution in sterile sea water was carried out with the soil sample and plated on Starch caesin agar prepared with filtered sea water. The growth media was supplemented with the antibiotics cycloheximide (25 mg/ml) and nalidixic acid (25 mg/ml) (Himedia, India). The seawater was filtered through 0.45 micron membrane filters (Millipore, India). For the isolation of actinobacteria pour plate technique was employed. The plates were incubated at 28° C for 2 to 4 weeks and were observed for the growth of actinobacterial colonies. The colonies were recognized according to their cultural characteristics and then transferred to slant culture at 4° C as well as at 20% (v/v) glycerol stock at -80° C for further use.
Extraction and purification of the lead compound from the isolate
From the well grown slant culture of the potential isolate VITDDK3, 106 spores/ml of the medium was inoculated into 250 ml Erlenmeyer flask containing 50 ml of production medium (Kuster’s broth with seawater) and the pH was adjusted to 9.0+0.2. The flask was incubated for 3 days in rotary shaker (150 rpm) at 28° C. From the seed culture, 5% of the inoculum was transferred into 100 ml of the production medium in 500 ml (10 Nos.) of Erlenmeyer flasks. The inoculated cultures were incubated for 7 days on a rotary shaker (150 rpm) at 28° C. After fermentation, the broth was centrifuged at 10,000 rpm for 10 min at 10o C. The supernatant was collected, separated by filtering through 0.2 micron membrane filter. The supernatant was extracted with equal volume of ethyl acetate. The upper ethyl acetate layer separated using separating funnel and concentrated in rotary vacuum and weighed. The crude compound of 250 mg was subjected to preparative TLC over silica plate with Chloroform: MeOH (1: 9, 1: 8, 2: 8 and 3: 8) as eluent. The single spot obtained on the TLC was scrapped and it was repeated for 10 times, finally pooled and dissolved in water. The supernatant was collected and subjected to silica gel column chromatography and eluted with Chloroform: MeOH (1:9). The fractions collected were pooled, concentrated and screened for antidermatophytic activity (separately for each eluent ratio). The active fraction was collected, concentrated and further checked for purity of the compound was evaluated by high performance liquid chromatography (HPLC; Perkin Elmer). The sample (20 µl) was injected into the silica packed (C-18) column and eluted with methanol (HPLC grade) pre-filtered using 0.22 µ membrane filter. The structure of the pure compound was established as reported earlier (Deepika et al., 2012).
Cell lines and cell culture
VERO (green monkey kidney) cell line, HEp-2 (Laryngeal cancer lines), HepG2 (Hepatocellular carcinoma), MCF7 (Human breast adenocarcinoma) and HT-29 (Human colon adenocarcinoma) cell lines were used for this study. Monolayer culture (48 h) of VERO, HEp-2, HepG2, MCF7 and HT-29 cell lines at a concentration of one lakh cells/well were seeded in 24 well titer plate. The plates were microscopically examined for confluent monolayer, turbidity, toxicity and whether the cells become confluent.
The cytotoxic effect of lead compound was tested by using MTT assay. The growth medium, Minimum Essential Medium (MEM) was removed using micropipette. The monolayer of cells was washed twice with MEM without Foetal calf serum (FCS) to remove the dead cells and excess FCS. To the washed cell sheet, 1ml of medium (without FCS) containing defined concentration of the drug in respective wells was added. Each dilution of the compound ranges from 1:1 to 1: 64 and they were added to the respective wells of the 24 well titer plates. To the control wells containing cells 1ml MEM without FCS was added. The plates were incubated at 37° C in 5 % CO2 environment and observed for cytotoxicity using inverted microscope.
After incubation of HTMO, the medium from the wells was carefully removed for MTT (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay. Each well was washed with MEM (without) FCS for 2-3 times and 200 µl of MTT (5 mg/ml) was added. The plates were incubated for 6-7 hr in 5% CO2 incubator for cytotoxicity. After incubation 1 ml of DMSO was added in each well, mixed and left for 45 sec. Viable cells present in the medium formed formazan crystals and it was dissolved by adding solubilizing reagent (DMSO) which resulted in formation of purple color. The absorbance of the suspension was measured spectrophotometrically at 595 nm by taking DMSO as a blank (Van and Viljoen, 2002).
Cell viability (%) = (Mean OD/Control OD) x 100
The effect of different concentrations of HTMO (84 µM, 168 µM, 336 µM, 672 µM, 250 µg/ml and 5263 µM) on different cancer cell line was also studied. The cell viability was measured after 24 h.
VITDDK3 were found to be gram positive, non-acid fast, non-motile, aerobic actinobacteria and produced no endospores. The aerial mycelia were branched; white in color and the substrate mycelium was pale yellow color (Fig.1 A). The isolate produced long chain of 10-25 spores with spherical in shape. The mature spores were 0.5-1.0 mm in diameter and the length was in between 0.8 and 1.0mm. Under scanning electron microscopy, the aerial mycelium of the isolate VITDDK3 were observed as unfragmented, branched, looped hyphae showing 2 curves and bearing non-motile spores with smooth surface (Fig.1 B).
Figure1. Streptomyces sp. VITBRK1 A) Culture in SCA media B) Scanning electron microscopic image showing the spore chain morphology and spore surface. The bar represents 10 μm.
Figure 2. Effect of HTMO on morphological changes of cancer cell lines. A) HT-29 control cells. B) HTMO (5263 µM) treated HT-29 cancer cells. C) HepG2 control cells. D) HTMO (5263 µM) treated HepG2 cancer cells. E) MCF7 control cells. F) HTMO (5263 µM) treated MCF7 cancer cells. G) HEp-2 control cells. H) HTMO (5263 µM) treated HEp-2 cancer cells. I) Normal VERO cells J) HTMO (5263 µM) treated VERO cells.
Figure 3. Effect of different concentrations of HTMO on cancer cell lines (HT-29, HEPG2, MCF7, HEp2) and normal VERO cell line.
The effect of different concentrations of HTMO on cancer cell lines is shown in Fig. 3. The HT-29 cells were more susceptible to HTMO at 1000 µg/ml concentration and VERO cells were least susceptible to HTMO at 1000 µg/ml concentration. MTT assay was carried out in 96 well plates with various concentrations of HTMO. The effect of HTMO on cancer cells and on Vero cells is shown in Table 1. The observed IC50 value indicated that HTMO was less toxic to the normal cells. HTMO was found to be cytotoxic and antiproliferative in nature on all the tested cell lines.
Table 1: Cytotoxic activity (IC50) of HTMO against selected cell lines determined by MTT assay
Values mean of 3 independent experiments
HTMO extracted from Streptomyces sp. VITDDK3 exhibited cytotoxic activity against tested cancer cell lines. Several cytotoxic compounds isolated from Streptomyces species were reported in the literature. A cytotoxic derivative isolated from New Zealand sponge Hymeniacidon hauraki (P-388) showed the IC50 value of 13.4 µg/ ml (Prinsep et al., 1994). 3,6-disubstituted indoles from Streptomyces sp. BL-49-58-005 was found to be cytotoxic to cancer cells (Sánchez López et al., 2003). Trioxacarcins from Streptomyces sp. B8652 showed anticancer activity (Maskey et al., 2004). Chartreusin extracted from Streptomyces chartreusi exhibited antitumor activity (Xu et al., 2005).
Piperazimycin A isolated from Streptomyces species exhibited potent in vitro cytotoxicity against multiple tumour cell lines (Miller et al., 2007). Allophycocyanin isolated from marine Streptomyces spp. M097 was reported to be an active peptide that inhibited the S-180 carcinoma in mice (Hou et al., 2006). Marine Streptomyces strains (PDK2, PDK7) isolated from South Indian coast, produced L-asparaginase and exhibited cytotoxic effect on JURKAT cells (Acute T cell leukemia) and K562 cells (chronic myelogenous leukemia) (Dhevagi and Poorani, 2006). Streptokordin, a new cytotoxic compound (methylpyridine class) was isolated from the culture broth of Streptomyces spp. KORDI-3238 (Jeong et al., 2006) and its cytotoxicity against several human cancer cell lines was also reported. Tetracenomycin D extracted from Streptomyces corchorusii AUBN1/ showed cytotoxic activity (Adinarayana et al., 2006). Selina-4(14),7(11)-diene-8,9-dio isolated from Streptomyces sp. QD518 demonstrated anticancer activity (Wu et al., 2006). Piericidins from Streptomyces sp. possess anticancer activity (Hayakawa et al., 2007).
Streptochlorin from Streptomyces sp. 04DH110 possess cytotoxic activity (Jae et al., 2007). 15-Hydroxy-T-muurolol from Streptomyces sp. M491 was reported to be cytotoxic (Ding et al. 2008). A novel furan types cytotoxic metabolite (HS071) isolated from Streptomyces spp. HS-HY-071 exhibited the IC50 value of 18.2 µg/ ml (Wang et al., 2008). Pyrroloiminoquinone from Streptomyces sp. CNR-698 exhibited cytotoxic activity (Hughes et al. (2009). Caboxamycin benzoxazole from Streptomyces sp. NTK 937 exhibited anticancer activity (Hohmann et al. 2009). Five isoflavone aglycones were isolated from culture filtrates of marine Streptomyces spp. 060524 isolated from the South China Sea and identified as genistein, glycitein and daidzein. The isoflavone glycoides showed strong cytotoxicity against K562 human chronic leukemia (Hu et al., 2009).
Three new cytotoxic 3,6-disubstituted indoles A, B and C obtained from Streptomyces spp. BL-49-58-005 were reported to be associated with Mexican marine invertebrate (Jose et al., 2003). Polyether antibiotics produced by Streptomyces spp. has been shown to possess broad spectrum of bioactivity ranging from antibacterial, antifungal, antiparasitic, antiviral and tumor cell cytotoxicity (Rutkowski and Brzezinski, 2013). Bioactive compounds 1,2-benzenedicarboxylic acid, bis (2-methyl propyl) ester and isooctyl phthalate extracted from Sreptomyces avidinii strain SU4 were reported to exhibit cytotoxic activity against Hep-2 cell line (Sudha and Masilamani, 2012). It was reported that the pigment extracted from Streptomyces sp. PM4 exhibited potential anticancer activity against HT1080, HE p-2, HeLa and MCF-7 cell lines with the IC50 value of 18.5, 15.3, 9.6 and 8.5 µg/ml respectively (Karuppiah et al., 2013). HTMO exhibited cytotoxicity on all the cancer cells tested. Among the cells tested HT-29 cells were highly susceptible to HTMO with IC50 value of 336 µM, followed by HepG2 (IC50: 672 µM), MCF7 (IC50: 672 µM), HEp-2 (IC50: 2688 µM) and on VERO cells it showed the IC50 value of 2688 µM. HTMO could be explored as a cytotoxic drug against cancer cells.
Authors are thankful to the management of VIT University for providing facilities to carryout this study.
Conflict of Interest
Authors declare that there is no conflict of interest.
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