Experimental Study Of The Active Ingredients In Traditional Chinese Medicine On Cell Migration Inhibition And Reversal Of Multidrug Resistance

Hepatocellular carcinoma (HCC) is a common malignancy in China and seriously harmful to human health. HCC is easy to metastasis and invasion which is important biological behavior of hepatoma cells. HCC prones to metastasis, which is an important factor of clinical outcomes, and the prognosis poor.Matrix metalloproteinases (MMPs) is a great family, named for their requirement of Ca2+, Zn2+ and other metal ions as a cofactor. His family members have a similar structure, and damage the normal structure through degrading extracellular matrix, thereby facilitating migration, invasion and metastasis of tumor cell. Tissue inhibitor of metalloproteinase (TIMP) is a specific inhibitor of MMPs, which can form complex with MMPs by covalent, specificly inhibit the activity of MMPs and degrade it, and thus inhibit the tumor metastasis.Ginsenoside, natural ingredients, extracted from ginseng, which is a steroid compounds, mainly existed in herbs of Panax ginseng. Ginsenosides is the active ingredient of ginseng and become the target of studies. The formula of ginsenoside Rh2 is C36H62O8H2O, and it molecular mass is 622. Previous studies showed that it can inhibit the proliferation of tumor cells from multiple links and promote the apoptosis of tumor cells. According to Tang’s report, ginsenoside Rh2 can inhibit invasion and metastasis of tumor in a certain extent, but the mechanism of this action is not completely understood. To further explore the intrinsic link between ginsenosides and invasion or metastasis of tumor, HepG2 cell as the experimental subjects of this study, preliminarily studied the influence of Rh2 on proliferation and migration of HepG2 cell and its mechanism, which can provide some experimental basis to clinical application.ObjectiveTo study the mechanism of ginsenoside Rh2 inhibiting HepG2 cell migration.MethodsHepG2 cells in logarithmic growth phase were cultured in 96-well plates, which were induced by different concentration of Rh2, respectively for 24,48,72 hours. The Cell proliferation activity was detected by Cell Counting Kit. Transwell chambers were used to check the migration ability of HepG2 cell; Luciferase was tested by Luciferase Reporter Assay system reagent; The expression of API, MMP3 fluorescence protein were observed by fluorescence microscopy; The expression of API, MMP3 gene were detected by Quantitative PCR; The expressions of P-ERK, ERK, P-P38, P-38, P-JUK, JUK, MMP3 proteins were detect by Western blot.Results1. Administrated with different concentration of Rh2 after 24,48,72 h, the proliferation of HepG2 cells were inhibited (P<0.05), and in dose-and time-dependent manner.2. Transwell assay showed Rh2 can significantly inhibited migration of HepG2 cells.3. Luciferase Report Gene Screened out AP1 transcription factor;4. AP1, MMP3 were distributed in the cytoplasm, the fluorescence intensity decreased with the increase of Rh2 concentration;5. Expression of AP1 and MMP3 genes were significantly decreased;6. The expressions of P-ERK, MMP3 proteins were significantly decreased, the expressions of P-JUK、P-P38 proteins were significantly increased, the expression levels of ERK, P-38, JUK were no significant difference.ConclusionGinsenoside Rh2 could inhibit the migration of HepG2 cells. Its mechanism may be through the MAPK pathway to inhibit API transcription factors, and then reduce the expression of MMP3.Leukemia is one of the highest incidences of malignant tumors in China, mostly cured by chemotherapy and bone marrow transplantation. The remission rate of leukemia has been greatly improved due to improving methods of combination chemotherapy, strengthening support therapy and applying hematopoietic stem cell transplantation. Chemotherapy is one of the primary treatments of leukemia, due to congenital resistant and acquired drug resistance is the main reason for the failure of chemotherapy.Evodiamine (EVO), dehydroepiandrosterone -Evodiamine and Rutaecarpine were extracted from Evodia, which belong to indole alkaloids. Pharmacological experiments showed that Evodia has an obvious effect on cell damage, antibacterial, antihypertensive, analgesic and uterine contractions. Foreign studies found that EVO had effect of anti-invasive and antimetastatic on a variety of tumor cell lines, including inhibitions of melanoma cells, cervical cancer cells, and prostate cancer cells. Long found that EVO could reverse the resistance of the resistant lung cancer cell line A549/DDP and increase the sensitivity of resistant to DDP. However, whether EVO can reverse the resistant of K562 cells, what is the mechanism? We made the following studies.ObjectiveTo research the effects of EVO on the proliferation, cell cycle and multi-drug resistance of leukemic K562 cells and its drug resistant strain. To probe the possible mechanisms of EVO reversing the multidrug resistance of K562 cell from the aspects of gene and protein.MethodsK562 and K562/Adr cells in logarithmic growth phase induced by EVO or/and DNR. The proliferation was detected by CCK-8 kit. Cell cycles were tested by flow cytometry; Flow cytometry was used to check the fluorescence intensity of DNR; Distribution of DNR was observed by fluorescence microscopy. The expression of MDR1 gene was detected by q-PCR. The expression of MDR1 and BCRP proteins were detected by western blot.Results1. CCK-8 test results showed that the proliferation of K562 and K562/Adr cells induced by EVO and DNR were inhibited in dose and time maner.2. The multiple drug resistance of DNR on K562/Adr cells was 30.54, the multiple drug resistance of EVO on K562/Adr cells was 19.09, compared with K562 cells; the reversal fold of DNR+EVO on K562/Adr cells was 12.07.3. FCM results showed that, in DNR+EVO induced groups the proportion of G2/M and S phase was significantly increased; the proportion of G0/G1 phase was reduced;4. FCM results showed the ratio of fluorescence was increased in the EVO+DNR group. The fluorescence enhancement in K562/Adr cells induced by DNR+EVO was more significant.5. Fluorescence microscopy showed that fluorescence intensity in the K562 and K562/Adr cell increased along with time; at the same reaction time, the fluorescence intensity of combination group was stronger than DNR group.6. PCR results showed that the expression of MDR1 gene were decreased in K562/Adr cells induced by EVO and DNR, but the expression of MDR1 gene in K562 cells had no significance changes.7. Western blot test results showed that the expression of BCRP and MDR1 protein of K562/Adr cells were significantly decreased; The expression levels of MDR1 protein in K562 cells were decreased either, but the expression level of BCRP protein were slightly decreased.ConclusionEvodiamine (EVO) can effectively reverse the multidrug resistance in K562/Adr cells and achieved by reducing the expression of MDR1 on cell membrane.