Saturday, 21 October 2017
A new clerodane furano diterpene glycoside from Tinospora cordifolia triggers autophagy and apoptosis in HCT-116 colon cancer cells
Journal of Ethnopharmacology Volume 211, 30 January 2018, Pages 295–310 Cover image Neha Sharmaa, b, Ashok Kumarc, f, P.R. Sharmac, f, Arem Qayumc, f, Shashank K. Singhc, f, Prabhu Dutta, Satya Pauld, Vivek Guptae, M.K. Vermab, N.K. Sattia, , , , R. Vishwakarmaa a Natural Product Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Jammu 180001, India b Analytical Chemistry Division (Instrumentation), CSIR‐ Indian Institute of Integrative Medicine, Jammu 180001, India c Cancer Pharmacology Division, CSIR‐ Indian Institute of Integrative Medicine, Jammu 180001, India d Department of Chemistry, University of Jammu, Jammu 180006, India e Post- Graduate Department of Physics, University of Jammu, Jammu 180006, India f AcSIR: Academy of Scientific and Innovative Research, Jammu- Campus, Jammu, India Received 5 July 2017, Revised 15 September 2017, Accepted 24 September 2017, Available online 27 September 2017 Show less https://doi.org/10.1016/j.jep.2017.09.034 Get rights and content Abstract Ethnopharmacological relevance Tinospora cordifolia is a miraculous ayurvedic herb used in the treatment of innumerable diseases such as diabetes, gonorrhea, secondary syphilis, anaemia, rheumatoid arthritis, dermatological diseases, cancer, gout, jaundice, asthma, leprosy, in the treatment of bone fractures, liver & intestinal disorders, purifies the blood, gives new life to the whole body; (rejuvenating herb) and many more. Recent studies have revealed the anticancer potential of this plant but not much work has been done on the anticancer chemical constituents actually responsible for its amazing anticancer effects. This prompted us to investigate this plant further for new potent anticancer molecules. Aim of the study The present study was designed to isolate and identify new promising anticancer candidates from the aqueous alcoholic extract of T. cordifolia using bioassay-guided fractionation. Materials and methods The structures of the isolated compounds were determined on the basis of spectroscopic data interpretation and that of new potent anticancer molecule, TC-2 was confirmed by a single-crystal X-ray crystallographic analysis of its corresponding acetate. The in vitro anti-cancer activity of TC-2 was evaluated by SRB assay and the autophagic activity was investigated by immunofluorescence microscopy. Annexin-V FITC and PI dual staining was applied for the detection of apoptosis. The studies on Mitochondrial Membrane potential and ROS (Reactive oxygen species) production were also done. Results Bioassay guided fractionation and purification of the aqueous alcoholic stem extract of Tinospora cordifolia led to the isolation of a new clerodane furano diterpene glycoside (TC-2) along with five known compounds i.e. cordifolioside A (β-D-Glucopyranoside,4-(3-hydroxy-1-propenyl)- 2,6-dimethoxyphenyl 3-O-D-apio-β-D-furanosyl) (TC-1), β-Sitosterol(TC-3), 2β,3β:15,16-Diepoxy- 4α, 6β-dihydroxy-13(16),14-clerodadiene-17,12:18,1-diolide (TC-4), ecdysterone(TC-5) and tinosporoside(TC-6). TC-2 emerged as a potential candidate for the treatment of colon cancer. Conclusion The overall study on the bioassay guided isolation of T.cordifolia identified and isolated a new clerodane furano diterpenoid that exhibited anticancer activity via induction of mitochondria mediated apoptosis and autophagy in HCT116 cells. We have reported a promising future candidate for treating colon cancer. Graphical abstract fx1 Figure options Abbreviations DAPI, 4′-6-Diamidino-2-phenylindole; MMP, Mitochondrial membrane potential; ROS, Reactive oxygen species; PTPC, permeability transition pore complex; PCD, Programmed cell death; MDC, Monodansylcadaverine; UV visspec, Ultra violet-visible spectrophotometer; IR, Infra-red; FTIR, Fourier Transform Infra-Red; LCMS, Liquid Chromatography-Mass Spectrometry; MeOH, Methanol; EtOAc, Ethyl acetate; CAN, Acetonitrile; TLC, Thin Layer Chromatography; CHCl3, Chloroform; DEPT, Distortionless enhancement by polarization transfer; HMBC, Heteronuclear Multiple Bond Correlation; NOESY, Nuclear Overhauser Effect SpectroscopY; TCA, Trichloroacetic acid; SRB, Sulforhodamine B; OD, Optical density; PBS, Phosphate buffer saline; DCFH-DA, dichlorodihydro-fluorescein diacetate) Keywords Tinospora cordifolia; DAPI (4′-6-Diamidino-2-phenylindole); Apoptosis; HCT116; MMP potential; ROS 1. Introduction Research on herbal drugs has stimulated multifold during the last few decades with special emphasis being on investigating novel candidates to combat existing life threatening diseases. Tinospora cordifolia is a miraculous ayurvedic herb used in “Rasayanas” for boosting immunity and body's strife against various infecting organisms ( Tirtha, 2005). It is globally distributed in Bangladesh, South Asia, Indonesia, Myanmar, Thailand, Sri Lanka, Pakistan, Nepal, in certain parts of China and throughout India ascending up to an altitude of 1200 m above sea level (Khare, 2007). Apart from more than 100 vernacular names such as Guduchi, Amrit (Sanskrit) and Abb-e-Hyat (Urdu) meaning water of life, it is also known as Giloya, a traditional term that alludes to the heavenly panacea which gives new life to the whole body, increases the human life span and keeps them perpetually young ( Sinha et al., 2004; Singh et al., 2003a ; Singh et al., 2003b). Giloya is used for curing innumerable health conditions such as diabetes, cancer, gonorrhea, secondary syphilis, irregular fever, skin diseases, anaemia, gout, rheumatoid arthritis, general and cardiac debility, cough, vomiting, chronic diarrhoea, asthma, in the treatment of bone fractures, liver & intestinal disorders, quite effective against lethal conditions like Swine Flu, Dengue and Malaria. It also purifies the blood and gives new life to the whole body; hence called as the rejuvenating herb (Singh, 1983; Shah, 1984; Bhatt, 1987; Shah et al., 1983; Kritikar, 1975; Zhao et al., 1991; Mhaiskar et al., 1980). The plant is used in numerous ayurvedic preparations such as Amritashtakachurana, Dashmoolarishta, Sanjivanivati, Kanta-kariavleha, GuduchyadichurnaChyavanprasha, Guduchisaatva, BrihatGuduchitaila, Guduchitaila, Stanyashodhanakashayachurana, Punehnimbachurna, Guduchighrita, Amrita guggulu, etc (Sinha et al., 2004). Traditionally, as per Indian Ayurveda it has been used in cancer treatment (Balachandran and Govinrajan, 2005; Williamson, 2002); local application of guduchi extracts was done to cure various tumors (Dash and Kashyap, 1987). In northern part of Gujarat in India, tribal people of Khedbrahma region consume the powdered root and stem/bark of this plant with milk for treating cancer (Bhatt, 1987). Juice and powder of the stem possesses anticancer effect in case of throat cancer ( Chauhan, 1995a ; Chauhan, 1995b) and breast cancer ( Chauhan, 1995a ; Chauhan, 1995b). Recently guduchi extracts have shown interesting results in experimental In vivo metastasis ( Leyon and Kuttan, 2004), inhibiting skin carcinogenesis (Chaudhary et al., 2008), antineoplastic effects in In vivo studies on Ehrlich ascites carcinoma ( Jagetia and Rao, 2006b) and cytotoxic effects in HeLa cells (Jagetia and Rao, 2006a). It's a well-known authentic immunomodulatory herb and is involved in helping the immune system in understanding the nature of cancer cells and ways to control them. It increases the immunity by promoting stem cell proliferation, elevating the levels of immunoglobulins and those of white blood cells. Toxicity study results have proven it to be non-toxic and with negligible side effects (Prakashananda, 1992). Guduchi powder/ Guduchi satva or Giloy Satva has exhibited promising effects on cancer which are either similar or even better than doxorubicin, a renowned chemotherapy drug which is used in the treatment of almost all cancers but is also associated with a number of side effects including cardiac myopathy in the later years after the treatment. 58.8% of reduction in the tumour volume by guduchi powder is quite comparable to cyclophosphamide which is another widely used anticancer drug (Sohini and Bhatt, 1996; Kapil and Sharma, 1997; Mathew and Kuttan, 1999). These remarkable properties of this herb can be utilized in cases where immunosuppression is mediated by cancer and hence could be a promising future drug of choice against various cancers, one such being colon cancer which is the third most common cause of cancer-related death and it is expected that in 2017, more than 90,000 new cases are to occur in the United States (U.S.) Medicinal value of any herb is directly related to the nature of the chemical constituents present in it. Extensive evidence based research done on the chemical constituents responsible for the various medicinal properties of this plant has revealed that myriad of biologically active phytoconstituents like alkaloids, tannins, phenolics, cardiac glycosides, flavanoids, sesquiterpenoids, saponins and steroids isolated from different parts of the plant have been attributed to the various pharmacological activities of Tinosporacordifolia ( Sudha et al., 2011; Rout, 2006; Ahmed et al., 2006; Kiem et al., 2010; Fukuda et al., 1993; Singh et al., 2005 ; Meghna et al., 2008; Jeychandran et al., 2003; Kumar et al., 2016a ; Kumar et al., 2016b; Grover et al., 2000; Premnath et al., 2010; Singh et al., 2005; Singh et al., 2003a ; Singh et al., 2003b; Kumar et al., 1981; Khuda et al., 1964; Sarma et al., 2009; Chi et al., 1994;Bisset et al., 1983;Khan et al., 1989). On the basis of the recent researches done, many articles have been cited to state the fact that the said plant has been used for treating various cancers. But as far as its anticancer chemical constituents are concerned, not much work has been done on this plant. Since it's a proven fact that the major breakthroughs in cancer drug discovery have been owed either to the natural products or the natural product scaffolds; vincristine, vinblastine, podophyllotoxin, taxol are few to be quoted. These discoveries- inspired by traditional and folk medicine clearly gives an indication that natural products are the future source for lead structures, and these will be used as templates for the development of more promising novel compounds with improved biological properties. Therefore, it is essential to use the available traditional knowledge and investigate the active plant extracts for the isolation of new, less toxic and highly efficacious molecules. Thus, in the course of our ongoing research and in a bid to arrive at more potential anticancer molecules, we investigated the bioactive aqueous fraction of Tinospora cordifolia via bioassay guided isolation and landed up with a new clerodane furano diterpenoid (TC-2) with potent activity against HCT-116 cells along with six known molecules. Apoptosis – an organized cell death causes specific morphological changes and cell death. However, exaggerated apoptosis and the impairment in the cell cycle can result in aberrant ability of multiplication in cancer cells. Thus, induction of apoptosis can be a treatment for cancer by reducing accumulation of cancer cells. Autophagy, the programmed cell death type-II also plays a part in tumour suppression ultimately self- destruction of cells in the body. Thus, the initiation of autophagic cell death via potent molecule can be an important approach for cancer prevention (Cotter, 2009). To ascertain whether the isolated new molecule, TC-2 induced cytotoxicity in HCT-116 cells could be the result of cell apoptosis, we examined the induction effect of this molecule on cell apoptosis of colon cancer cells, HCT-116 by DAPI staining, annexin-V/PI dual staining and on mitochondrial membrane potential studies. To investigate whether TC-2 also caused autophagy in HCT-116 cells, it was examined by monodansylcadaverine (MDC) staining by fluorescence microscopic studies and also detection of LC3b was carried out by the immunofluorescence confocal microscopic studies. 2. Materials and methods 2.1. General experimental procedures, chemical and biochemicals Buchi, B-542 was used for recording the melting points. UV spectras were measured with Shimadzu UV-2600 UV–Vis Spectrophotometer and the IR spectras were obtained with Perkin Elmer FT-IR, Spectrum Two. 1H and 13C NMR data was recorded on Bruker-Advance DPX FT NMR 500 and 400 MHz instruments. LCESIMS data was acquired on Agilent UHD-6540 LCMS/MS (HRMS) system. Silica gel of #60–120 and #100–200 mesh size from Merck Germany, SupelcoDiaion HP-20SS, U.SA, Sephadex and LH-20 from Sigma Aldrich were used for chromatographic separation, isolation and purification of the compounds. 2.2. Plant material Fresh stems of Tinospora cordifolia were procured from Indian Institute of Integrative Medicine (IIIM), Jammu, India. A voucher specimen (RJM/0010) was identified by the botany division and deposited in the herbarium of the Institute. 2.3. Cell culture, growth conditions and treatment conditions Human lung carcinoma cell line (A549), Prostate (PC-3), SF-269 (CNS), MDA-MB-435 (Melanoma), HCT-116 (Colon) and Breast (MCF-7) were procured from NCI: National Cancer Institute, USA. Culturing of the cancer cells was done in RPMI-1640 medium consisting of 10% FCS, penicillin (100 units/ml) and streptomycin (100 μg/ml). Standard culture conditions were employed. The cell cultures were grown in CO2 incubator (New Brunswick, Galaxy 170 R, Eppendorf)) at37 °C with 98% humidity and 5% CO2 gas environment. 2.4. Extraction and isolation Fresh stems (1.5 kg) of Tinospora cordifolia were crushed, soaked in 4.5 L of ethyl acetate: water in the ratio of 1:1 and mechanically stirred for 2 h. The mixture was double filtered through muslin cloth and distilled on rota vapour at 55 °C. 59.2 g of the aqueous portion (coded as TCE) obtained was freeze dried. Freeze dried extract was divided into two parts, 20 g of which was used for column chromatography on Diaion HP-20 (400 g, column size - 90 cm × 6 cm). The column was eluted successively with 100% water (9 × 500 ml), 25% MeOH/ H2O (6 × 500 ml), 50% MeOH/ H2O (4 × 500 ml), 100% MeOH (15 × 500 ml) and 50% EtOAc/MeOH (3 × 500 ml). A total of 47 fractions, each of 500 ml volume were collected. Fractions 11–14 yielded 125 mg of residue with one major spot on the T.L.C. On repeated column chromatography of the pooled 11–14 fractions on Sephadex LH-20, 14 mg of white amorphous compound was obtained which was identified as cordifolioside A (coded as TC-1) on the basis of spectral data obtained with that reported in the literature. 560 mg of fraction 20–22 (coded as TCFR) was taken up for further purification for flash chromatography on RP-18 silica gel (33.5 g). Column was eluted with water (3 × 100 ml), 10% ACN in water (2 × 100 ml), 20% ACN in water (2 × 100 ml), 30% ACN in water (2 × 100 ml), 40% ACN in water (2 × 100 ml), 50% ACN in water (2 ×100 ml), 100% ACN (2 × 100 ml), 100% MeOH (3 × 100 ml) and finally with isopropyl alcohol. On the basis of similar TLC pattern obtained, fraction no. 8-12 (TCFR-3) from a total of 30 fractions were pooled and dried. 121 mg of the residue so obtained was re-chromatographed on RP-18 silica gel. Elution with 100% ACN and repeated crystallization in methanol led to the isolation of 76 mg of creamish colored pure compound, coded as TC-2. Remaining 39.2 g form the freeze-dried extract dissolved in water was extracted with butanol and further used for isolation. Butanol extract was chromatographed over silica gel column (100–200 mesh, column size – 90 cm × 15 cm) and eluted with a combination of 1–100% MeOH/CHCl3 with 112 fractions. Initial fractions eluted in chloroform yielded octacosanol, TC-3 (10 mg) followed by the isolation of β-sitosterol, TC-4 (25 mg). Fractions 33–39 (eluted with 10% MeOH/CHCl3) and 48–56 (eluted with 15% MeOH/CHCl3), crystallized in methanol and acetone afforded clerodadiene-diolide, TC-5 (15 mg) and Ecdysterone, TC-6 (10 mg). 82–91 of the total 112 fractions on repeated column chromatography yielded Tinosporaside, TC-7 (32 mg) and crystallized in acetone respectively. The identification of the compound structures isolated from the column were determined on the basis of the spectroscopic data with that reported in the literature. All the collected fractions were subjected to preliminary screening for their anticancer activity in order to identify the bioactive fractions for the isolation of potent anticancer molecules. 2.5. Structure elucidation 2.5.1. Spectral analysis TC-2 was obtained as creamish white amorphous solid. Analysis of the 13 C NMR and DEPT-135 spectra revealed 28 resonances along with a pseudo molecular ion peak [M-H3O]+ at m/z 605. Its IR spectrum showed absorptions corresponding to hydroxyl and furan moieties at 3384 cm−1 and 771.62 cm-1. Signals at 1729.05 cm-1 indicated the presence of lactone which was confirmed by the presence of resonance at 172.062 ppm in the 13 C NMR spectrum. The UV absorption at 209 nm supported the presence of an α, β-unsaturated ketone group. 1H-NMR of TC-2 displayed signals at δ 7.42 (t, 1H, J = 3.3 Hz), δ 7.43 (d, 1H, J = 0.6 Hz) and δ 6.40 (dd, 1 H, J = 0.9 Hz) suggested the presence of protons of the β-substituted furan moiety commonly reported in clerodanes isolated from different Tinospora species. One angular methyl group at C-9 was observed as three proton singlet at 1.09 respectively. The signals at δ 3.40 (m) were assigned to the C-12 proton bearing the β-substituted furan moiety. The signals at the aliphatic region δ 1.94 (m) and δ 1.74 (m) were attributed to the C-3 methylene protons (Fig. 1, 2 and 3; Table 1). Fig. 1 Fig. 1. 1H NMR of compound TC-2. Figure options Fig. 2 Fig. 2. 13C NMR of compound TC-2. Figure options Fig. 3 Fig. 3. DEPT-135 spectral images of compound TC-2. Figure options Table 1. NMR Data for TC-2 in CDCl3 (δ in ppm, J in Hz in parentheses), bs- broad signal. Position δC δH HMBC Correlation observed in 1 COSY NOESY 1 76.31 3.41 m – 3.4(C-12) 2 70.47 3.53 m – 3 30.49 1.94b, 1.74a m – I)2.4(C-11),2.10(3′,4′)-II)2.2(C-7) 4 75.51 3.31 m – 3.57(C-2) 5 34.58 Q – – 6 25.17 1.74a,1.447b m – 7 21.39 2.36a,1.73b m – 8 51.10 2.31 m – CH3-9 9 35.14 Q – – 10 39.06 1.67 m CH3-9, 5′, 4′, 2′ 11 43.53 2.42a,1.73b d at 2.42(3.25) & m at 1.73 – 12 73.28 3.40 m – 3.54(C-1), 4.27(C-1′) 13 125.38 Q – – 14 108.16 6.40 d,1H, (0.9) C-13,C-15,C-16 15 139.22 7.44 d,1H, (0.6) C-13,C-14,C-16 6.4(C-14) 16 143.901 7.43 in HSQC t,1H,(3.3) C-13,C-15,C-14 17 172.062 Q – – 1′ 99.91 4.26 d,H, (7.7 Hz) – 3.38(C-4) 2′ 68.61 5.46 dd,1H,(3.15) and (2.95) – 3′ 71.02 5.61 dd, 1H, (3.10) and (3.20) – 1.76(C-3 or C-6) 4′ 73.37 4.96 dd, 1H, (3.25) and (3.25) – 5′ 73.79 3.84 m, bs – 6′ 62.30 3.83b,3.78a m, bs – 9-CH3 21.53 1.09 s,3H C-11,C-8,C-2 1.65 (C-10) H8, H10 2′-OCOCH3 21.06 1.94 s, 3H – 3′-OCOCH3 20.94 2.13 s, 3H – 2.42 (C-11), 4′-OCOCH3 21.147 2.36 s, 3H – 2′-OCOCH3 172.06 Q – – 3′-OCOCH3 170.60 Q – – 4′-OCOCH3 169.57 Q – – Table options The HMBC correlations observed from H-14 (δ 6.4) to C-13 (δ 125.38), C-15 (δ 139.22) & C-16(δ 143.90); H-15 (δ 7.44) to C-13 (δ 125.38), C-14 (δ 108.16) & C-16(δ 143.90); H-16 (δ 7.43) to C-13 (δ 125.38), C-14 (δ 108.16) & C-15 (δ 139.22) and CH3-9 (δ 1.09) methyl protons to C-2 (δ 70.47), C-8 (δ 51.10) and C-11 (δ 43.53) augmented the above argument (Fig. 4a and Fig. 4a ; Fig. 4bb). Fig. 4a Fig. 4a. Major HMBC correlations between protons of C-14, 15 & 16 with Carbons 13, 14,15 & 16 in compound TC-2. Figure options Fig. 4b Fig. 4b. Major HMBC correlations (blue arrows) for compound TC-2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Figure options Of the three hydroxyl resonances, the one at δ 3.10 was allocated to the carbon at position 1. The other deshielded hydroxyl peak at δ 3.53 was assigned to the C-2 based on HMBC correlations. The relative configuration of TC-2 was deduced based on NOESY experiment. A cross peak was found from H-8 (δ 2.31) to CH3-9 (δ 1.09); H-10 (δ 1.67) to CH3-9 (δ 1.09), H-5′ or 6′ (δ 3.83), H-2′ (δ 5.46), H-4′(δ 4.96); CH3-9 (δ 1.09) to H-8 (δ 2.31) and H-10 (δ 1.67) indicated similar configurations between different groups which was confirmed by the X-ray crystallography. Thus TC-2 was assigned the following structure (Fig. 7). HPLC chromatogram and Mass spectrum of TC-2 has been represented in Fig. 5 and 6. The other compounds from TC-1, TC-3 to TC-7 were identified on the basis of comparing their spectral data with the ones already reported in the literature (Supplementary data: S1-S20). Fig. 5 Fig. 5. HPLC chromatogram of TC-2. Figure options Fig. 6 Fig. 6. Mass spectra of TC-2. Figure options Fig. 7 Fig. 7. Chemical structure of TC-2. Figure options 2.6. Acetylation of TC-2 TC-2 was reacted with acetic anhydride in catalytic amount of pyridine to obtain acetate crystals of the isolated molecule for X-ray structure determination and confirmation. 10 mg of TC-2 was dissolved in 1 ml of pyridine and 2 ml of acetic anhydride was added to the solution. The reaction mixture was heated on a steam bath for 2 h under dry conditions. Usual work up followed by crystallization yielded triacetate, TC-2acetate (Fig. 8). In 1H NMR (500 Hz, CDCL3), signal shifts from δ 3.41, 3.53, 3.84 and 3.778 to δ and increase in number of 9 protons at δ 2.1 confirmed the formation of three acetate groups (Fig. 9). The triacetate so formed was confirmed by X-ray crystallography. Fig. 8 Fig. 8. Molecular structure of TC-2 acetate with atomic labelling. Figure options Fig. 9 Fig. 9. 1H NMR of compound TC-2acetate. Figure options 2.7. X-ray crystal studies of TC-2 2.7.1. Crystal structure determination and refinement X-ray intensity data of 7288 reflections (of which 4004 unique) were collected on X′calibur CCD area-detector diffractometer equipped with graphite monochromated MoKa radiation (λ = 0.71073 Å). The dimensions of the TC-2 acetate crystal used were 0.30 × 0.20 × 0.20 mm. The intensities were measured by w scan mode for q ranges 3.55–26.00. 1149 reflections were treated as observed (I > 2σ(I)). Data were corrected for Lorentz, polarization and absorption factors. The structure was solved by direct methods using SHELXS97 (Sheldrick, 2008). All non-hydrogen atoms of the molecule presented the best E-map locations. Full-matrix least-squares refinement experiment was performed using SHELXL97 (Sheldrick, 2008). The final refinement cycles converged to an R = 0.0614 and wR(F2) = 0.1204 for the observed data. Residual electron densities ranged from −0.175 < Δρ < 0.409 eÅ-3. Atomic scattering factors were referred from International X-ray Crystallography tables (1992, Vol. C, Tables 220.127.116.11 and 18.104.22.168). The crystallographic data has been summarized in Table 2. The molecular structure of the TC-2 acetate with atomic labelling is shown in Fig. 8. Table 2. Crystallographic data of TC-2 acetate. CCDC no. 1545869 contains the crystallographic data. The data can be obtained free of cost via www.ccdc.cam.ac.uk/data_request/cif by e-mailing data request @ ccdc.cam.ac.uk, or by contacting The Cambridge Crystallography Data Centre, 12 Union Road, Cambridge, CB2 IEZ, UK. Fax: +44(0) 1223-336033. Computer programs: SHELXL97 (Sheldrick, 1997). CCDC deposition No. 1545869 Chemical formula C36H46O17 Mr 750.73 Temperature (K) 293 a, b, c (Å) 12.4621 (15), 7.4751 (6), 21.069 (3) α, β, γ (deg.) 90, 105.277 (13), 90 V (Å3) 1893.3 (4) Z 2 Radiation type Mo Kα µ (mm−1) 0.11 No. of measured, independent and observed [I > 2σ(I)] reflections 7288, 4004, 2173 Rint 0.049 (sin θ/λ)max (Å−1) 0.617 Refinement R[F2 > 2σ(F2)], wR(F2), S 0.061, 0.147, 0.99 No. of reflections 4004 No. of parameters 486 No. of restraints 1 H-atom treatment H atoms treated by a mixture of independent and constrained refinement Δρmax, Δρmin (e Å−3) 0.41, −0.18 Absolute structure Flack H D (1983), Acta Crystal. A39, 876–881 Absolute structure parameter 10 (10) Table options 2.7.2. Crystal packing The packing of the molecules in the unit cell is shown in Fig. 10. From the figure, it is evident that the molecules related by twofold screw are packed in layers. The stability of the crystal structure was due to the the presence of O-H···O and C-H···O intra- and intermolecular hydrogen bonds. Details of O–H···O and C–H···O hydrogen bonds are given in Table 3 and those details on the bond lengths and bond angles of the non-hydrogen bonds has been provided in the Supplementary section; S21-S23. Fig. 10 Fig. 10. The packing arrangement of molecules in TC-2 acetate, viewed down the b-axis. Figure options Table 3. Geometry of intermolecular hydrogen bonds in TC-2 acetate. D-H…A D-H (Å) D…A(Å) H…A(Å) D-H…A,(deg.) C8-H18-O3 0.98 3.495(7) 2.59 153 C33-H39B-O12 0.96 3.436(8) 2.50 165 C31-H42A…O7 0.96 3.136(11) 2.57 118 Symmetry codes: (i) -x+1, y+1/2,-z (ii) –x+z, y-1/2, -z+1 (iii) x+1, y-1, z. Table options An ORTEP view of TC-2 with atomic labelling and the unit cell packing view of the TC-2 molecules down the b-axis is shown in Fig. 10 (Farrugia, 2012) and Fig. 11. The molecular geometry was analyzed and calculated using the WinGX (Farrugia, 1997), PLATON (Spek, 2009) and PARST (Nardelli, 1995) softwares. Fig. 11 Fig. 11. ORTEP view of molecule TC-2 acetate with displacement ellipsoids drawn at 40%. H atoms are shown as small spheres of arbitrary radii. Figure options 2.8. In vitro cytotoxicity assay Sulforhodamine B (SRB) testing was performed, in which cell suspension of cell density 7500−15,000 cells/100 μL was seeded. Cultures were incubated with 1 µM, 10 µM, 30 µM and 50 µM concentrations of test material in complete growth medium (100 μL) after 24 h of incubation. Paclitaxel, mitomycin, 5-fu and doxorubicin were used as positive controls. After further 48hr incubation, cells were fixed with ice-cold TCA for 1hr at 4 °C. Plates were washed atleast five times with distilled water (D.W) and kept for air drying. Further 100 μL of 0.4% sulphorhodamine-B (SRB) solution added into each well of the dried plates was allowed for 30 min staining at room temperature. SRB solution was detached by quickly washing of the plates with 1% v/v acetic acid in order to remove the excess unbound dye. The bound SRB dye was solubilised by adding 100 μL of 10 mM unbuffered Tris Base (pH 10.5) to each well and with continuous shaking for 5 min on a shaker platform to solublize the dye completely, and finally the reading was taken at 540 nm on microplate reader (Thermo Scientific). IC50 was determined by plotting OD against concentration from Graph PAD Prism version 5. 2.9. Nuclear morphology studies by DAPI staining The presence of apoptotic cells was ascertained by staining human colon cancer HCT-116 cells with DAPI. Seeded HCT-116 cells (2 × 105/ml/well) in 60 mm culture dishes. After 24 h, cells were incubated with various concentrations of TC-2 and paclitaxel (positive control) for 24 h. Media was collected and cells were rinsed with PBS. Trypsinization was done in order to detach the cells followed by their back addition to the conditioned media to ascertain the incorporation of the floating and poorly attached cells in the analysis. Air dried smears of HCT-116 cells were fixed in methanol at −20 °C for 20 min, air dried and stained with DAPI at 1 µg/ml in PBS at room temperature for 20 min in the dark and the slides were placed in glycerol-PBS (1:1) and examined in an inverted fluorescence microscope (Olympus, 1X81) (Rello et al., 2005) 2.10. Detection of apoptosis by Annexin V-FITC and PI Annexin V-FITC and propidium iodide (PI) dual staining technique is generally employed to detect the early and late stages of apoptosis (PCD type 1). For evaluating apoptosis, HCT-116 cells were splitted and made confluent in six-well plates (2 × 105 cells) and treated with TC-2 for 24 h and paclitaxel (1 μM) was used as a positive control. 24 h after treatment, cells were collected, washed with PBS and suspension was made in binding buffer. Following that, staining of the cells was done with Annexin V/FITC and PI for 15 min in dark and studied by laser scanning confocal microscope using appropriate lasers (Olympus Fluoview FV 1000) (Zhang et al., 2013; Bai et al., 2015; Dai et al., 2008; Munafo et al., 2001; Acharya et al., 2009;Kumar et al., 2016a ; Kumar et al., 2016b) 2.11. Detection of intracellular reactive oxygen species (ROS) accumulation Intracellular ROS levels were examined by fluorescence microscopy after staining with DCFH-DA (dichloro dihydro-fluorescein diacetate). HCT-116 cells (2 × 105/ml/well) were seeded in 60 mm culture dishes and after 24 h, were incubated with different concentrations of TC-2 for 24 h and examined under fluorescence microscope using 40X lens (Olympus, 1X81). H2O2 (0.05%) was used as positive control (Bai et al., 2015; Kumar et al., 2016a ; Kumar et al., 2016b). 2.12. Loss of mitochondrial membrane potential (MMP) Loss in mitochondrial membrane potential (∆ψm) as a result of mitochondrial disturbance was examined using confocal microscopy after staining with Rhodamine 123 (Rh123). Human colon cancer (HCT-116) cells (2 × 105/ml/well) were seeded in six well plate and treated for 24 h with different concentrations of TC-2 and paclitaxel (positive control). Cells untreated and treated with test materials were trypsinized and washed twice with PBS. Thereafter cell pellets were then suspended in fresh medium (2 ml) containing Rh123 (1.0 μM) and incubated at 37 °C for 20 min with gentle shaking. Following that, the cells were centrifuged, collected and washed twice with PBS, then examined by laser scanning confocal microscope (Olympus Fluoview FV1000) (Dai et al., 2008; Kumar et al., 2016a ; Kumar et al., 2016b) 2.13. Analysis of autophagy by monodansylcadaverine (MDC) staining MDC, Monodansylcadaverine is a prescribed marker for labelling of autophagic vacuoles. HCT-116 cells (2 × 105/ml/well) were seeded in 60 mm culture dishes and after 24 h, were incubated with different concentrations of TC-2 for 24 h. MDC labelling of the autophagic vacuoles was done with by incubating the cells for 1 h with 0.05 mM MDC in PBS at 37 °C. After incubation, cells were washed thrice with PBS and forthwith examined by fluorescence microscope using 40X lens (Munafo et al., 2001;Kumar et al., 2016a ; Kumar et al., 2016b). 2.14. Immunofluorescence microscopic studies for detection of cytochrome C and LC3B HCT-116 cells (2 × 105/ml/well) were seeded in 60 mm culture dishes and after 24 h, were incubated with different concentrations of TC-2 for 24 h. MDC labelling of the autophagic vacuoles was done with by incubating the cells for 1 h with 0.05 mM MDC in PBS at 37 °C. After incubation, cells were washed thrice with PBS and forthwith examined by fluorescence microscope using 40X lens (Munafo et al., 2001;Kumar et al., 2016a ; Kumar et al., 2016b). HCT-116 cells were seeded over cover slips in 35 mm culture dishes, incubated with different concentrations of TC-2 for 24 h. Cells were washed two times in PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Forthwith 10 min permeabilization of the cells in 0.1%TritonX-100/PBS at room temperature was done. Cells were incubated in 10% BSA to block the nonspecific binding sites followed by their incubation with primary antibodies cytochrome c and LC3B diluted 1:100 in 0.1% Triton X-100 in PBS for 1hr at room temperature, followed by washing with PBS and incubation with respective Alexa Fluor 555and 488 conjugated secondary antibodies diluted 1:500 in PBS for 1 h at room temperature in dark. Cells were then washed thrice in PBS and stained with DAPI (1 µg/ml) in PBS. The cover slips were mounted over glass slides and images of cells were taken using a laser scanning confocal microscope (Olympus Fluoview FV1000) employing 60X oil immersion objective lens (Acharya et al., 2009; Kumar et al., 2016a ; Kumar et al., 2016b) 3. Results Out of all the fractions from the aqueous extract of Tinospora cordifolia evaluated for their cytotoxicity against different cancer cell lines, TCFR fraction was found to be more active than the other fractions, Table 4a. The bioactive fractions were subjected to repeated column chromatography for the expected isolation of more potent molecules. All the isolated molecules were screened for their anticancer potential where TC-2 exhibited more promising results than the other molecules. Using SRB assay, we evaluated the cytotoxicity of TC-2 on a panel of human cancer cell lines of various origin for a period of 48 h incubation. The following cell lines were used: Lung (A549), Prostate (PC-3), SF-269 (CNS), MDA-MB-435 (Melanoma), HCT-116 (Colon) and Breast (MCF-7). From the screening experiments, compound TC-2 was found to be most active on Prostate (PC-3), MDA-MB-435 (Melanoma) and HCT-116 (Colon) cancer cell lines. To determine the IC50 values, cells were treated with different concentrations of the compound. The results showed that the incubation of different cancer cells with 1 µM, 10 µM, 30 µM and 50 µM concentrations of TC-2 for 48 h imparted varied effects on cellular viability. IC50 values were of the order of 8 µM (HCT-116) in colon carcinoma, 10.4 µM (PC-3) Prostate, 14.8 µM (MDA-MB-435) for melanoma, 23 µM (SF-295) in CNS carcinoma, 33 µM (A549) in lung cancer and 40 µM (MCF-7) in breast carcinoma, Table 4b. Table 4a. In vitro cytotoxicity of the extract, bioactive fraction and the isolated compounds against human cancer cell lines. Cell line type HCT-116 A549 PC-3 Tissue Colon Lung Prostate S.NO. Code Conc. (µg/ml) % Growth inhibition 1. T.C.E 50 23 0 27 100 52 5 48 2. TCFR-3 50 62 2 72 100 94 9 97 3. TC-1 50 0 12 5 100 20 19 14 4. TC-2 50 94 77 71 100 97 78 78 5. TC-3 50 3 11 26 100 9 25 43 6. TC-4 50 7 13 11 100 24 16 20 7. TC-5 50 12 0 0 100 27 2 10 8. TC-6 50 0 0 3 100 0 0 17 5-Fluorouracil 20 µM 52 – – Paclitaxel 1 µM – 76 – Mitomycin 1 µM – – 66 Table options Table 4b. In vitro cytotoxicity of TC-2 against 6 human cancer cell lines. TISSUE LUNG CNS PROSTATE MELANOMA COLON BREAST CELL LINE A549 SF-295 PC-3 MDA-MB-435 HCT-116 MCF-7 CODE CONC.(µM) %CYTOTOXICITY TC-2 1 0 0 0 23 0 0 10 16 41 51 42 57 0 30 45 49 62 69 88 20 50 73 92 78 86 99 74 IC50 33 23 10.4 14.8 8 40 Paclitaxel 1 77 < 0.01 – – – – Mitomycin-C 1 – – 63 – – – 5-FU 20 – – – – 52 – Doxorubicin 1 – – – – – 65 Table options To examine, whether TC-2 treatment killed cancer cells by inducing apoptosis, we analyzed the human colon cancer (HCT-116) cells for nuclear morphological changes, by staining nuclei with DAPI. TC-2 induced chromatin condensation and fragmentation of nuclei of few cells in concentration dependent manner, typical of apoptosis (Fig. 12). Annexin V/PI dual staining suggested the significant externalization of PS (phosphatidylserine) in the events of early cell death after 24 h in the present studies. The concentration dependent increase in percentage of early and late stage apoptosis with treatment of TC-2 was observed (Fig. 13). We evaluated mitochondrial membrane potential (MMP) changes with laser scanning confocal microscope which exhibited considerable loss of MMP in human colon cancer (HCT-116) cells with different concentrations of TC-2 (Fig. 14). Further, Cytochrome c localization was determined by immunofluorescence with a cytochrome c specific antibody. Cytochrome c was colocalized in mitochondria of untreated cells. In contrast, TC-2 and paclitaxel treatments displayed decrease in functional mitochondria and release of cytochrome c to the cytosol respectively (Fig. 15). Fig. 12 Fig. 12. Nuclear morphology analysis of HCT-116 cells (2 × 105/ml/well) using DAPI. After treatment with indicated concentrations of TC-2 for 24 h and examined using fluorescence microscopy (40X). Paclitaxel (1 μM) was used as positive control. With increase in concentration of TC-2 there is significant increase in nuclear condensation and formation of apoptotic bodies. Figure options Fig. 13 Fig. 13. The representative images of TC-2 treatment on the exposure of phosphatidylserine (PS) in HCT-116 cells after 24 h treatment. Phosphatidylserine exposure was assessed by the Annexin V/propidium iodide assay and analyzed by confocal microscopy using 40× oil immersion lens. Histogram showing the percentage of cells in early and late stages of apoptosis obtained by analysis of the cell images. Data are mean ± S.D. of three similar experiments; statistical analysis was done with *p < 0.05. Figure options Fig. 14 Fig. 14. Loss of mitochondrial membrane potential (∆ψm) was measured in human colon cancer (HCT-116) cells (2 × 105/ml/well) treated with indicated concentration of TC-2 in 6 well plates for 24 h and incubated with Rodamine-123 (1.0 µM) in serum free media for 20 min at 37 °C and washed with PBS. The loss of mitochondrial membrane potential (MTP) in HCT-116 cells was observed under laser scanning confocal microscope using 40X lens (Olympus Fluoview FV1000). Figure options Fig. 15 Fig. 15. Colocalization of cytochrome c and mitochondria was determined by confocal microscopy using 60× oil immersion lens. Human colon cancer (HCT-116) cells were immunostained for cytochrome c release (green) and the mitochondria of the cells were stained with MitoTracker (red). HCT-116 cells were treated with different concentrations of TC-2 and paclitaxel (1 µM) for 24 h and stained with anticytochrome c antibody and Alexa Fluor 488-labeled secondary antibody. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.). Figure options TC-2 increased ROS/oxidative stress in HCT-116 cells in concentration dependent manner (Fig. 16). We also ascertained changes in autophagic activity by examining the fluorescence of MDC, which has been known as a specific marker for autophagic vacuoles. The number of autophagic vacuoles stained by MDC in the TC-2 treated HCT-116 cells was much higher than in the untreated cells (Fig. 17). Next, to further confirm the induction of autophagy by TC-2, a set of autophagy- related factors including LC3-I and LC3-II in the HCT-116 cells after treatment with different concentrations of TC-2 for 24 h were studied by immunofluorescence microscopy. The microtubule associated protein light chain 3 (LC3) is an additional signature marker of autophagosomes. Cleavage of the 18 kDa full length LC3, known as LC3-I, to a 16 kDa form, known as LC3-II, results in recruitment of LC3-II to double layered membrane of autophagosomes and this is a key step in autophagy. The immunofluorescence confocal microscopic studies of TC-2 treated HCT-116 cells too displayed concentration dependent induction of autophagy (Fig. 18). Fig. 16 Fig. 16. Intracellular ROS level was detected by fluorescence microscopy using 2′,7′- dichlorofluorescein diacetate (DCFH-DA) after 24 h treatment. HCT-116 cells were treated with indicated concentrations of TC-2 and 0.05% H2O2 and incubated with 5 μM 2′,7′-dichlorofluorescein diacetate and examined by fluorescence microscope (40X). Figure options Fig. 17 Fig. 17. TC-2 induces autophagy in HCT-116 cells. The autophagic vacuoles were observed under fluorescence microscope (40×) with MDC staining. The treatment of compound and BEZ235 (positive control group) induced concentration-dependent formation of autophagic vacuoles in HCT-116 cells after 24 h. Figure options Fig. 18 Fig. 18. Detection of autophagy with LC3b antibody by confocal microscopy using 60X oil immersion lens. Immunocytochemical staining was conducted using anti-LC3b antibody and Alexa Flour-555-labeled secondary antibody. Nuclei were stained with DAPI. Figure options 4. Discussion TC-2 revealed highest activity against colon cancer (HCT-116) cells and least activity against breast cancer (MCF-7) cells with IC50 values of 8 and 40 μM respectively. To understand the interesting potency revealed by this new clerodane diterpenoid, detailed mechanistic studies were carried out. The differential cytotoxicity exhibited by the compound may be due to the varying molecular characteristics of these cells. Additionally the differential cytotoxicity of TC-2 against different human cancer cell lines exhibits that its use against different types of cancers might present promising results. These findings substantiate the findings in Polyalthia longifolia ( Verma et al., 2008) and Ocimum basilicum ( Manosroi et al., 2006). The property of cancer cells is their resistance to apoptosis induction (Hanahan et al., 2000). Therefore, inducing apoptosis is the aim of many anticancer therapeutic approaches as it makes it possible to kill cancer cells without causing inflammation (Reed, 2002). Mitochondria are intermediate to the intrinsic apoptotic pathway and thus are important targets for curing cancer (Fulda et al., 2010). Mitochondrial membrane permeabilization (MMP) and release of pro-apoptotic proteins (e.g. cytochrome c) from the intermembrane space to the cytosol are the characteristic features of the mitochondrial pathway of apoptosis. These events lead to the activation of the initiator caspase 9, thereby triggering the caspase cascade causing DNA condensation/fragmentation and ultimately cell death (Kroemer et al., 2009). MMP involves pore formation such as Bax/Bak oligomers and the permeability transition pore complex (PTPC) (Kroemer et al., 2007). Mitochondria plays a major role in energy generation and are also significant sensors for apoptosis. ROS is generated during cellular metabolism through leakage of electrons by mitochondrial electron transport and is a mediator in apoptosis. To gain insight into the mechanism by which TC-2 results in cell death we next examined ROS production, since excessive generation of ROS results in cell injury and death. Mitochondria play a significant role in apoptosis. Mitochondria-mediated reactive oxygen species (ROS) generation is a major source of oxidative stress in the cells. The apoptosis induction in the present studies causes the activation of the mitochondrial pathway and is subsequently associated with ROS production, Cyt c release, and nuclear fragmentation. The other modes of non-apoptotic cell death by plant-derived anti-cancer drugs are also emerging, and mainly comprise autophagy etc. In the current study, TC-2 also induces autophagy in HCT-116 cells. The present studies are in conformity with the earlier findings wherein the synthesized natural alkaloid berberine derivatives have also been reported to induce autophagy in human colon carcinoma HCT-116 and SW613-B3 cells (Guaman et al., 2015). Similarly natural compounds have also been reported to induce autophagy, such as rottlerin (Torricelli et al., 2012), chrysin-organotin based on chrysin (Xuan et al., 2016), betanin/isobetanin (Nowacki et al., 2015), cucurbitacin B (Ren et al., 2015). In conclusion, we demonstrated that the new clerodane diterpenoid, TC-2 induces apoptosis of colon cancer (HCT-116) cells mainly by triggering ROS production. This new natural compound thus shows potential for the treatment of colon cancer. Autophagy was also observed after the treatment. Taken together, our study identified a new clerodane furano diterpenoid that exhibited anticancer activity via induction of mitochondria mediated apoptosis and autophagy in HCT116 cells. The results from the present studies will be very advantageous in the further development of new chemotherapeutic agents. Acknowledgements Two authors, Neha Sharma and Ashok Kumar are highly thankful to the Department of Science and Technology, New Delhi for the award of INSPIRE fellowship. Acknowledgements Author's contribution NKS and MKV conceived and designed the study. NS carried out the bioassay guided isolation of the new molecule along with six known compounds from T.cordifolia. Compounds were characterized by NKS, MKV and NS. PD conducted the purity profile of the isolated molecules. via HPLC.VG carried out the X-ray crystallographic experiments and interpreted the crystal data. AQ and SKS conducted the preliminary screening of the extracts and the molecules on different cancer cell lines. AK and PRS carried out the detailed mechanistic study of the new clerodane diterpenoid on colon cancer cell lines. SP and RAV helped in drafting the manuscript. 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