R-HepG2 Cells Research Articles

Polyphyllin D - A Potential Anti-Cancer Agent to Kill Hepatocarcinoma Cells with Multi-Drug Resistance

Abstract: To develop drugs to kill cancer cells, we chemically synthesized a number of anti-cancer agents by adding dif-ferent side chains to the core backbone of saponin. With the use of bioassay-guided methods, we found one agent that possessed a high cytotoxicity to a number of cancer cell lines. Interestingly, this compound was later found to be an active component of a tradition Chinese herb Paris polyphylla known as Polyphyllin D (PD) (diosgenyl -L-rhamnopyranosyl-(1
2)-(-L-ara-binofuranosyl-(1
4)--D-glucopyranoside). In China, the rhizome of Paris polyphylla (Chong Lou) has been used as a traditional Chinese medicine to treat a number of cancers including pancreas and liver cancers for a long time. Results from our laboratory demonstrate that PD is a potent anti-cancer agent that bypasses multi-drug resistance (MDR) and induces programmed cell death in R-HepG2 cells over-expressing P-glycoprotein (P-gp). In this paper, we re-viewed the mechanisms how PD overcomes the MDR and exhibits a stronger cytotoxicity in the R-HepG2 than its parent line without P-gp through mitochondrial injury.

Anti-Proliferative effect of ursolic acid on multidrug resistant hepatoma cells R-HepG2 by apoptosis induction

Many studies have demonstrated that ursolic acid (UA) could potently inhibit the proliferation of a wide variety of cancer cells; however, its anti-tumor effect on multidrug resistant carcinoma was rarely reported. In the present study, UA exhibited cytotoxicity on the multidrug resistant human hepatoma cell line R-HepG2 with the IC50 value of 20 μM at 48 h. Our findings indicated that UA was not a binding substrate for P-glycoprotein and could not modulate the expression and activity of P-glycoprotein. This provides a possible mechanism on UA circumvention of the drug resistance. Mechanistic study demonstrated that UA induced apoptosis on R-HepG2 cells via both extrinsic and intrinsic apoptotic pathways, including the activation of caspase cascade and the release of cytochrome c and AIF from mitochondria to cytosol. Furthermore, in vivo study showed a significant inhibition on the growth of R-HepG2 cells in the UA-treated group (15 mg/kg/day, i.v.), with negligible damage towards the heart and the liver. The present study suggests the potential of UA as chemotherapeutic agent and provides further insight into the molecular basis of UA-induced apoptosis on multidrug resistance human hepatoma cells.

Alteration of mitochondrial membrane potential by Spirulina platensis C‐phycocyanin induces apoptosis in the doxorubicinresistant human hepatocellular‐carcinoma cell line HepG2

C-PC (C-phycocyanin) is a water-soluble biliprotein from the filamentous cyanobacterium Spirulina platensis with potent antioxidant, anti-inflammatory and anticancerous properties. In the present study, the effect of C-PC was tested on the proliferation of doxorubicin-sensitive (S-HepG2) and -resistant (R-HepG2) HCC (hepatocellular carcinoma) cell lines. These studies indicate a 50% decrease in the proliferation of S- and R-HepG2 cells treated with 40 and 50 microM C-PC for 24 h respectively. C-PC also enhanced the sensitivity of R-HepG2 cells to doxorubicin. R-HepG2 cells treated with C-PC showed typical apoptotic features such as membrane blebbing and DNA fragmentation. Flow-cytometric analysis of R-HepG2 cells treated with 10, 25 and 50 microM C-PC for 24 h showed 18.8, 39.72 and 65.64% cells in sub-G(0)/G(1)-phase respectively. Cytochrome c release, decrease in membrane potential, caspase 3 activation and PARP [poly(ADP-ribose) polymerase] cleavage were observed in C-PC-treated R-HepG2 cells. These studies also showed down-regulation of the anti-apoptotic protein Bcl-2 and up-regulation of the pro-apoptotic Bax (Bcl2-associated X-protein) protein in the R-HepG2 cells treated with C-PC. The present study thus demonstrates that C-PC induces apoptosis in R-HepG2 cells and its potential as an anti-HCC agent.

Pheophorbide a, an active component in scutellaria barbata, reverses P-glycoprotein-mediated multidrug resistance on a human hepatoma cell line R-HepG2

Scutellaria barbata, a Traditional Chinese Medicine native in southern China, has been widely used for treating liver diseases. In this study, the anti-proliferative effect of Pheophorbide a (Pa), an active component from S. barbata, was examined on a multi-drug resistant (MDR) human hepatoma cell line R-HepG2. Our study showed that Pa could significantly inhibit the growth of R-HepG2 cells with an IC50 value at 25.0μM after 48 hours treatment. When compared with the parental HepG2 cells, Pa showed weak resistance to R-HepG2. Efflux of Pa out of R-HepG2 cells was not observed as its cellular uptake level showed no significant difference comparing with HepG2 cells. Interestingly, significant reduction of P-glycoprotein expression on Pa-treated R-HepG2 cells was found at both transcriptional and translational levels, leading to reduction of P-glycoprotein activity. In addition, mechanistic study elucidated that Pa induced cell cycle arrest at G2/M phase and inhibited the expressions of G2/M phase cell cycle regulatory proteins, cyclin-A1 and cdc2 in a dose-dependent manner.

Riboregulator H19 induction of MDR1-associated drug resistance in human hepatocellular carcinoma cells

Acquisition of drug resistance is one of the main obstacles encountered in cancer chemotherapy. Overexpression of multi-drug resistance 1 (MDR1) gene and its protein product P-glycoprotein, accompanied with a decrease in doxorubicin accumulation level, was observed in doxorubicin-resistant R-HepG2 cells, a subline derived by selection of human hepatocellular carcinoma HepG2 cells with doxorubicin. In addition, Northern-blot analysis revealed an eight fold upregulation of the imprinted H19 mRNA in R-HepG2 cells. H19 knockdown by transfection with antisense H19 oligonucleotides suppressed the MDR1/P-glycoprotein expression, increased the cellular doxorubicin accumulation level and sensitized doxorubicin toxicity in both HepG2 parent cells and R-HepG2 cells. Results from methylation-specific polymerase chain reaction analysis indicated that the MDR1 gene promoter was hypomethylated in R-HepG2 cells. Antisense H19 oligonucleotides transfection induced a marked increase in the percentage of MDR1 promoter methylation and decrease in MDR1 expression in R-HepG2 cells. Thus, the H19 gene is believed to induce P-glycoprotein expression and MDR1-associated drug resistance at least in liver cancer cells through regulation of MDR1 promoter methylation.

Anti-cancer activity and mechanisms of 25-anhydrocimigenol-3-O-??-D-xylopyranoside isolated from Souliea vaginata on hepatomas

Our previous study first revealed the cytotoxicity and relative selectivity of 25-anhydrocimigenol-3-O-beta-D-xylopyranoside (ACX) on HepG2 and R-HepG2 cells. In the present study, the anti-cancer activity and mechanisms of ACX isolated from S. vaginata were investigated both in vitro and in vivo. ACX showed significant, consistent anti-proliferative activity on hepatoma bel-7402 cells by MTT and clone formation assays with an IC50 value of 18 mumol/l. Morphological observation and flow cytometry results showed that apoptosis and G0/G1 cell cycle arrest contributed to the cytotoxic and cytostatic effects. Further studies showed that Bax and p21 protein expression were upregulated, Bcl-2 protein expression was downregulated, and poly(ADP-ribose) polymerase protein was cleaved. Moreover, ACX also exhibited a dose-dependent inhibition of tumor growth on mice implanted with H22 in vivo. These findings implicate ACX as a promising anti-cancer agent for chemotherapy of certain cancers.

Anti-hepatoma activity and mechanism of ursolic acid and its derivatives isolated from Aralia decaisneana

To investigate the anti-tumor activity of ursolic acid (UA) and its derivatives isolated from Aralia decaisneana on hepatocellular carcinoma both in vitro and in vivo. In vivo cytotoxicity was first screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Morphological observation, DNA ladder, flow cytometry analysis, Western blot and real time PCR were employed to elucidate the cytotoxic mechanism of UA. Implanted mouse hepatoma H22 was used to evaluate the growth inhibitory effect of UA in vivo. UA could significantly inhibit the proliferation of HepG2 and its drug-resistance strain, R-HepG2 cells, but had no inhibitory effect on primarily cultured normal mouse hepatocytes whereas all the six derivatives of UA could not inhibit the growth of all tested cell lines. Further study on mechanism demonstrated that apoptosis and G0/G1 arrest were involved in the cytotoxicity and cleavage of poly-(ADP-ribose)-polymerase (PARP). Downregulation of cyclooxygenase-2 (COX-2) protein and upregulation of heat shock protein (HSP) 105 mRNA correlated to the apoptosis of HepG2 cells treated with UA. In addition, UA also could inhibit the growth of H22 hepatoma in vivo. UA is a promising anti-tumor agent, but further work needs to be done to improve its solubility.

Cytotoxicity of cycloartane triterpenoids from aerial part of Cimicifuga foetida

Our previous study had demonstrated cytotoxicity of EtOAc fraction from aerial part of Cimicifuga foetida on various cancer cell lines. In the present study, we investigated the cytotoxicity of five cycloartane triterpenoids isolated from the EtOAc fraction. Compounds ( 1,2,3) showed moderate cytotoxic activity on R-HepG2 cells and drug resistant sub line R-HepG2 but exhibited less toxicity against primary cultured normal mouse hepatocytes. Compounds ( 4) and ( 5) were inactive on all tested cell lines.

Effect of arsenic trioxide on multidrug resistant hepatocellular carcinoma cells

Our previous study showed that arsenic trioxide (As 2O 3) was effective in inhibiting the growth of human hepatocellular carcinoma (HepG2) cells via induction of apoptosis. In the present study, we examined the effect of As 2O 3 on multidrug resistant human hepatocellular carcinoma (R-HepG2) cells which are characterized with overexpression of mdr1 gene and P-glycoprotein. The anti-proliferation of R-HepG2 by As 2O 3 was examined by MTT assay. For the induction of apoptosis, DNA fragmentation and Annexin V-PI staining were performed after treatment with arsenic trioxide. To study the effect of arsenic trioxide on P-glycoprotein, Western analysis probing anti-P-glycoprotein antibody was used to monitor the change of its expression. Results showed that As 2O 3 was effective in inhibiting the cell proliferation of R-HepG2 cells in a dose- and time-dependent manner via induction of apoptosis without affecting the cell cycle. The sensitivity of R-HepG2 cells toward As 2O 3 was found to be similar to that of the parental HepG2 cells. The Western analysis showed that As 2O 3 was probably not the substrate to be bound and extruded by P-glycoprotein in R-HepG2 cells because it could not maintain the cellular P-glycoprotein expression.

Cytotoxicity of three cycloartane triterpenoids from Cimicifuga dahurica

We first investigated the cytotoxicity of three cycloartane triterpenoids isolated from the aerial part of C. dahurica. Their cytotoxic activity was investigated on several cancer cell lines including solid tumor (HepG2), blood tumor (HL-60), drug resistant tumor (R-HepG2) and primary cultured normal mouse and rat hepatocytes in order to find efficient anti-tumor agents against both parental and drug-resistant tumor with reduced toxicity. Evident cytotoxicity of these compounds on all tested neoplastic cell lines revealed that they are efficient on both drug-resistant tumor and parental tumor. Furthermore, they all showed relatively selective cytotoxicity on cancerous cells based on the higher IC 50 values of them on normal cells than that on tumor cells. Morphological observation and cell cycle analysis were employed to elucidate the cytotoxicity of the tested compounds. They brought out similar apoptotic morphological changes and G 2/M cell cycle arrest in HepG2, R-HepG2 and HL-60 cells. Moreover, they suppressed the expression of cdc2 and COX-2 protein. These results imply that the three compounds possess potential anti-tumor activities and they exert their cytotoxicity via apoptosis and G 2/M arrest. In addition, inhibition of cdc2 protein expression correlates with mechanism of G 2/M arrest.

Polyphyllin D is a potent apoptosis inducer in drug-resistant HepG2 cells

In a search for new anticancer agents, we identified a novel compound polyphyllin D (PD) (diosgenyl α- l-rhamnopyranosyl-(1→2)-(α- l-arabinofuranosyl)-(1→4)]-[β- d-glucopyranoside) that induced DNA fragmentation and phosphatidyl-serine (PS) externalization in a hepatocellular carcinoma cell line HepG2 derivative with drug resistance (R-HepG2). PD is a saponin originally found in a tradition Chinese medicinal herb Paris polyphylla. It has been used to treat liver cancer in China for many years. We evaluated the cell-killing mechanisms of this compound in R-HepG2 and its parental cells. The mitochondrial apoptotic pathway was found to be involved in the PD-induced apoptosis because PD elicited depolarization of mitochondrial transmembrane potential (Δ Ψm), generation of H 2O 2, as well as release of cytochrome c and apoptosis-inducing factor in a dose- and time-dependent manner. In conclusion, we show for the first time that PD is a potent anticancer agent that can overcome drug resistance in R-HepG2 cells and elicit programmed cell death via mitochondrial dysfunction.

Mitochondria-Targeting Drug Oligomycin Blocked P-Glycoprotein Activity and Triggered Apoptosis in Doxorubicin-Resistant HepG2 Cells

Background: Mitochondria are key regulators in apoptosis. This suggests that a mitochondrion can be a target for cancer treatment. To examine the feasibility of this approach, we investigated the effect of oligomycin on the induction of apoptosis in drug-resistant cells. As a mitochondrion-targeting agent, oligomycin inhibits mitochondrial F₀F₁-ATPase. Of 37,000 molecules tested against the 60 human cancer cell lines of the National Cancer Institute, oligomycin is among the top 0.1% most cell line selective agents. Methods: Changes in the doxorubicin (Dox) accumulation and mitochondrial potential (Δψm) in human hepatocarcinoma HepG2 and its derivative R-HepG2 with Dox resistance were determined by flow cytometry. P-glycoprotein (Pgp) expression and release of cytochrome c from mitochondria were analyzed by Western blot. Cytotoxicity was examined by DNA fragmentation and the alamar blue assay. Results: R-HepG2 cells produced Pgp, showed drug resistance and accumulated less Dox when compared to their parent. In both cell lines, oligomycin depolarized Δψm, released cytochrome c and elicited DNA fragmentation. Moreover, oligomycin blocked Pgp activity and accumulated more Dox in R-HepG2. Combined treatment with Dox and oligomycin elicited more cell death. Conclusion: Our results suggest that oligomycin could bypass Dox resistance and trigger apoptosis in R-HepG2 cells.

Circumvention of multidrug resistance and reduction of cardiotoxicity of doxorubicin in vivo by coupling it with low density lipoprotein

Doxorubicin (Dox) was coupled into human low density lipoprotein (LDL) to form a complex LDL-Dox. In in vitro studies, the accumulation of LDL-Dox in human resistant hepatoma (R-HepG2) cells was found to be higher than that of free Dox in the cells, resulting in an increase of the cytotoxic effect on the cells. Moreover, in in vivo studies, under the same dosage of drugs (1 mg/kg), the anti-proliferative effect on the tumor cells of LDL-Dox in nude mice bearing R-HepG2 cells was higher than that of free Dox as evidenced by the larger reduction in tumor volumes and tumor weights in LDL-Dox treated group. Histological studies showed that LDL-Dox treatment did not cause any heart damage when compared with the control group. In contrast, Dox treatment caused disruption and vacuolization of myocardial filament. Plasma lactate dehydrogenase activity and plasma creatine kinase activity in nude mice bearing R-HepG2 cells were found to be elevated in the Dox-treated group but remained unchanged in LDL-Dox-treated group. The present studies indicate that when Dox is coupled with LDL, the multidrug resistance can be circumvented and the cardiotoxicity can be reduced.

Mitochondrial targeting drug lonidamine triggered apoptosis in doxorubicin-resistant HepG2 cells

Mitochondria play a crucial role in the induction and execution of apoptosis. Accordingly, recent suggestions have been made to use agents that directly act on mitochondria to trigger apoptosis so that drug-sensitive and-resistant tumour cells can be eliminated. To test this hypothesis, human hepatocarcinoma HepG2 and its derivative R-HepG2 with doxorubicin (Dox) resistance as a result of expression of P-glycoprotein were used to investigate the effect of lonidamine (LND), a new mitochondrial targeting drug, on the induction of apoptosis. Results from our study indicate that R-HepG2 cells were more sensitive to LND than parental cells in terms of cytotoxicity determined by alamar blue assay. Cell death induced by LND was associated with the hallmarks of apoptosis such as mitochondrial membrane depolarization, release of cytochrome c, phosphatidyl-serine externalization and DNA fragmentation. Moreover, combined treatment of cells with Dox and LND elicited more cell death. Taken together, our results suggest a potential use of LND as an anti-cancer drug to bypass drug resistance and to trigger tumour destruction through apoptosis in HepG2 and R-HepG2 cells.