[논문]Induction of Apoptosis by Methanolic Extract of Rubia Cordifolia Linn in HEp-2 Cell Line is Mediated by Reactive Oxygen Species

Shilpa, P.N.; Sivaramakrishnan, V.; Devaraj, S. Niranjali

doi:10.7314/apjcp.2012.13.6.2753

Induction of Apoptosis by Methanolic Extract of Rubia Cordifolia Linn in HEp-2 Cell Line is Mediated by Reactive Oxygen Species 원문보기

Asian Pacific journal of cancer prevention : APJCP, v.13 no.6, 2012년, pp.2753 - 2758

Shilpa, P.N. (Department of Biochemistry, University of Madras) , Sivaramakrishnan, V. (School of Chemical and Biotechnology, SASTRA University) , Devaraj, S. Niranjali (Department of Biochemistry, University of Madras)

Abstract ▼ AI-Helper

Rubia cordifolia Linn, which belongs to the Rubiaceae family, is a well-known herb used in Ayurvedic medicine. In the present study, we investigated the influence of a methanolic extract (RC) on the induction of apoptosis in HEp-2 (human laryngeal carcinoma) cell line, as evidenced by cytotoxicity, morphological changes and modification in the levels of pro-oxidants. Inhibition of cell proliferation and lactate dehydrogenase (LDH) release increased in a time and dose-dependent manner. Further, reduced glutathione (GSH), glutathione transferase (GST) and protein levels decreased and lipid peroxidation increased significantly on RC treatment in a dose dependent manner when compared to controls. Based on the results we determined the optimal dose as 30mg/ ml and the apoptotic effect of RC extract (30 mg/ml) on HEp-2 cells was confirmed by fluorescent microscopy and transmission electron microscopy (TEM) based on morphological and ultrastructural changes. RC extract suppressed the proliferation of HEp-2 oral cancer cells inducing apoptotic cell death in vitro. These results point to potential of RC extract as an agent for the treatment of laryngeal squamous cell carcinoma.

주제어

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가설 설정

Thus these concentration were chosen for morphological characterization which showed: (a) Viable cells with normal nuclei (VN)-Bright green chromatin with organized structure.(b) Viable cells with apoptotic nuclei (VA)-Bright green chromatin highly condensed or fragmented.(c) Non-viable cells with normal nuclei (NVN)-Bright orange chromatin with organized structure.
(c) Non-viable cells with normal nuclei (NVN)-Bright orange chromatin with organized structure. (d) Non-viable cells with apoptotic nuclei (NVA)-Bright orange chromatin, highly condensed or fragmented.

제안 방법

In this study, HEp-2 cells treated with RC in the dose range of 5, 10, 15, 20, 25, 30 mg/ml, lactate dehydrogenase (Figure 1b) release increased in a time and dose- dependent manner. GSH and GST levels decreased and lipid peroxidation increased significantly on RC treatment (Figure 1c, 1d, 1e).

대상 데이터

Human Epithelial carcinoma (HEp-2) cell line was obtained from National Center for Cell Sciences, Pune, India. The cells were grown at 37℃ under a humidified, 5% CO₂ atmosphere in DMEM medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 units/ml of penicillin and 50 μg/ml of streptomycin.

데이터처리

The results were statistically evaluated using one-way analysis of variance (ANOVA) by SPSS software version 10 for repeated measurements and Post-hoc Dunnet’s test for multiple comparisons was performed.

이론/모형

Lactate dehydrogenase (LDH) was estimated by the method of (Nieland, 1955). The activity of GlutathioneS-Transferase (GST) was determined by the method of (Habig et al.
, 1974). Reduced glutathione (GSH) was estimated by the method of (Moron et al., 1979). Lipid peroxide (LPO) content was determined by thiobarbituric acid reaction as described by (Okhawa et al.
Lactate dehydrogenase (LDH) was estimated by the method of (Nieland, 1955). The activity of GlutathioneS-Transferase (GST) was determined by the method of (Habig et al., 1974). Reduced glutathione (GSH) was estimated by the method of (Moron et al.

성능/효과

In conclusion, our result clearly demonstrated that RC inhibits cell proliferation in HEp-2 cell line and induced apoptosis through the elevation of reactive oxygen species generation. This could be attributed to phytochemicals present in the extract.
These assays clearly indicated that RC inhibited the proliferation of cancer cells. The results also showed that the antiproliferative activity of RC at the concentrations used in the study was completely due to killing and not by inhibition of cell division. The effect was found to be dose and time dependent.

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