Hep3B cells were treated with progressively increasing concentrations of Paclitaxel in cell culture medium for selection of Paclitaxel-resistant cells. After consecutive Paclitaxel treatments for a period of approximately 2 months, several numbers of drug resistant clones were developed from the Hep3B cell line. We termed those clone Drug Resistance Clone (DRC). Drug resistance clone (DRC) of Hep3B was used for all subsequent experiments in this study. In addition, we also developed a unique single cell clone (SCC) of Hep3B by plating diluted cell suspension and allowed to incubate until they made clones. We selected few clones and MTT assay was done and we observed that single cell clones also exhibited drug resistance. To compare the growth properties and cell cycle analysis of He3B and its derived clones DRC and SCC, we did not observed any noteworthy change in growth and cell cycle distribution (Fig. 1A). To determine the cell survival capacity of both Paclitaxel-sensitive Hep3B and Paclitaxel-resistant cells, Hep3B and its derived cones DRC and SCC cells were treated with 1 µM Paclitaxel for 48 hrs. Paclitaxel-sensitive Hep3B cells showed blebbing and cell rounding with empty spaces visualized surrounded by the cells. This suggested that a large fraction of cells were undergone in G2/M phase, with some of these cells undergoing apoptosis. On the other hand, no noticeable morphological change was observed in Paclitaxel-resistant DRC and SCC cells (Fig. 1B). Cell death was also observed by flow cytometry analysis after staining with propidium iodide (Fig. 1C). In cooperation with both assays detected a smaller percentage of apoptotic cells in Paclitaxel-resistant DRC and SCC, compared to their parental Hep3B cells after treatment with 1 µM Paclitaxel for 48 hrs (Fig. 1B and 1C). The protein expression of the cleaved Poly (ADP-ribose) polymerase (c-PARP), an important marker of caspase-mediated apoptosis, was also examined by Western blotting after the cells were treated with 1 µM Paclitaxel for 48 hrs. We observed much lower levels of cleaved PARP and correspondingly much higher levels of un-cleaved PARP in Paclitaxel-resistant DRC and SCC cells, compared to parental Hep3B cells (Fig. 1D). Cell viability assay showed that DRC and SCC cells could tolerate much higher concentrations of Paclitaxel compared to Hep3B cells, with their IC50 concentrations found to be more higher than Hep3B cells (Fig. 1E).
Increased expression of caveolin-1 in DRC and FASN and Cytochrome P450 in SCC Paclitaxel-resistant cells
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To examine the role of various proteins in mediating Paclitaxel resistance in human liver cancer cells, the expression of caveolin-1, Fatty acid synthase and cytochrome P450 and p-glycoproteins was examined in Hep3B, DRC and SCC cells. We found that a caveolin-1 and p-glycoproteins level was markedly increased in DRC only. However, FASN and cytochrome P450 level was increased in DRC as well as SCC cells, compared to their parental Hep3B cells (Fig. 2A). Cells or tissues from a extensive variety of tumors have been shown to express different levels of one or more Hsps. Such observations have led to suggestions that Hsps could be used as biomarkers. For example, Hsp expression in breast or gastric cancer is associated with poor prognosis and resistance to chemotherapy or radiation therapy. Heat shock proteins are also reported that they are playing a big role in drug resistance. We also checked whether heat shock proteins are also involved in resistance. We found that Hsp70, Hsp40, Hsp 90 and Hsp27 were not change in Hep3B and its derived clones. These results might be indicating that acquired Paclitaxel resistance is correlated with the increased caveolin-1 and p-glycoproteins expression in DRC. while in SCC which is inherently resistance have high level of FASN and cytochrome P450. Interestingly, treatment with Paclitaxel resulted in the induction of cavolin-1 expression in a dose-dependent pattern in Hep3B and SCC cells (Fig. 2C). To study the mechanism that may contribute to the increased expression of caveolin-1, Hep3B cells were treated with CHX to block protein synthesis and the cells were further treated with or without Paclitaxel for different times, the protein stability of caveolin-1 was measured by Western blot (Fig. 2D). The result showed that caveolin-1 protein is more stable in Paclitaxel treated cells than that of untreated cells. We further compared the mRNA level of caveolin-1 in Paclitaxel-treated and untreated cells by qRT-PCR (Fig. 2E). The result showed that Paclitaxel treatment increased the mRNA expression of caveolin-1. These results suggest that both protein stability and mRNA induction by Paclitaxel contribute to the up-regulation of caveolin-1 in these cells.
The downregulation of caveolin-1, FASN and cytochrome P450 re-sensitizes Paclitaxel resistance clones
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The increase of caveolin-1, FASN and cytochrome P450 expression and FASN activity detected in Paclitaxel-resistant cells suggests that caveolin-1 may play a critical role in Paclitaxel resistance in DRC and FASN in SCC. Therefore, the effect of Caveolin-1, FASN and cytochrome P450 downregulation on the sensitivity of Paclitaxel was investigated. Since the expression and activity of FASN was upregulated in Paclitaxel-resistant SCC cancer cells and caveolin-1 expression was upregulated in DRC (Fig. 2), we hypothesized that the downregulation of caveolin-1, FASN and cytochrme P450 by siRNA might re-sensitize Paclitaxel-resistant cells to Paclitaxel. To this end, caveolin-1 was knocked down with siRNA in DRC and parental Hep3B cells respectively, and then the cells were treated with different concentrations of Paclitaxel. The downregulation of caveolin-1 increased the sensitivity of these cells to Paclitaxel, with Paclitaxel-resistant DRC cells showing about a 3-10 fold increase in cell growth inhibition under 50 -100 nM Paclitaxel treatment measured by both MTT assay (Fig. 3C) and direct cell counting (Additional file 1, Figure. S1). Interestingly, DRC cells showed a much greater overall increased sensitivity to Paclitaxel compared to their parental Hep3B cells cells (Fig. 3C and 3D). Similar assays were performed in another SCC cell line by using FASN specific siRNA (Fig. 4 A-C), where the knockdown of FASN expression by siRNA increased the sensitivity to Paclitaxel by at least 2-fold. To further confirm these results, SCC cells with specific siRNA was used for knockdown of FASN expression and activity were used. Compared to those of control Hep3B cells, FASN expression (Fig. 4D) and FASN activity (Fig. 4E) were dramatically decreased in FASN knockdown cells and these cells showed a much greater overall increased sensitivity to Paclitaxel (Fig. 4F). These results demonstrated that caveolin-1 plays an important role in Paclitaxel resistance in DRC and FASN is crucial for Paclitaxel resistance in SCC.
The combination of Paclitaxel with Methyl β-cyclodextrin shows synergistic inhibitory effect on drug resistant clone (DRC)
The water-soluble methylated form MβCD is known to form soluble inclusion complexes with cholesterol, thereby enhancing its solubility in aqueous solution. MβCD is employed for the preparation of cholesterol-free products: the bulky and hydrophobic cholesterol molecule is easily lodged inside cyclodextrin rings that are then removed. We first examined the effect of MβCD on caveolin-1 expression and cell viability of Hep3B and DRC cells. MβCD treatment led to a decrease of caveolin-1 expression (Fig. 5A) and an inhibition of cell viability (Fig. 5B) in a dose-dependant manner, in DRC. Compared to Hep3B cells, Paclitaxel resistant DRC cells showed a greater sensitivity to MβCD, consistent with the results of caveolin-1 knockdown by siRNA (Fig. 3). These results support the notion that increased Paclitaxel sensitivity by MβCD is a consequence of the inhibition of membrane caveolin-1 and depletion of cholestrol. Since downregulation of caveolin-1 by siRNA or MβCD significantly inhibited the viability of the Paclitaxel-resistant cells, we further investigated the effects of combining Paclitaxel with MΒCD on Paclitaxel-resistant DRC. Paclitaxel combined with MβCD were much more effective in inhibiting cell viability compared with either agent given alone. Similar treatment combinations were performed SCC, but there was no significant difference was found in these inherent clone. Taken together, the combination of Paclitaxel with MβCD has a greater capacity to inhibit acquired drug resistant clone compared to either agent given alone. To further investigate the mechanism of MβCD-induced in acquired DRC Paclitaxel re-sensitization, we examined cellular apoptosis in these cells. PARP, a nuclear protein that can be easily cleaved by caspases, has been widely used as an apoptosis marker. The expression level of total PARP and cleaved PARP (c-PARP) were examined in DRC cells after treatment with Paclitaxel, MΒCD, or their combination for 48 hrs, respectively. We found a significant increase of the levels of cleaved PARP after treatment with the combination of Paclitaxel and MΒCD compared to treatment with single agent (Fig. 6D). This indicates that cellular apoptosis is a mechanism involved in the increased cell growth inhibitory effect of the combination treatment of Paclitaxel with MΒCD.
The combination of Paclitaxel with Cerulenin shows synergistic inhibitory effect on single cell clone (SCC)
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The increased de novo fatty-acid synthesis is caused by multiple mechanisms, including increased expression of lipogenic enzymes. Among them, FASN overexpression is observed in a wide variety of human cancers. FASN is not only overexpressed in cancer, but it also plays an essential role in tumour growth, survival and drug resistance. Hypoxia and low pH stress induce the FASN expression in cancer cells (Menendez et al, 2005; Furuta et al, 2008). Hypoxia upregulates SREBP-1, the major transcriptional regulator of the FASN gene, through phosphorylation of Akt Although the precise mechanisms of FASN inhibition-induced
cell death in cancer cells still remain unknown, several possibilities have been proposed. Initial studies indicate that FASN inhibition accumulates the toxic intermediary metabolite, malonyl-CoA, which induces apoptosis, whereas pharmacological inhibition of ACC by 5-(tetradecyloxy)-2-furoic acid does not (Pizer et al, 2000). In addition to the essential role of FASN in cancer cell growth and survival, it is involved in other phases of cancer development. Browne et al showed that Orlistat, an antiobesity drug, inhibits FASN and suppresses endothelial cell proliferation and angiogenesis, suggesting a novel role of endothelial cell FASN in vivo tumour growth (Browne et al, 2006). FASN overexpression also confers resistance to Adriamycin and mitoxantrone in breast cancer cells (Liu et al, 2008). These observations suggest that FASN inhibition could be a novel strategy to interfere with tumour survival through angiogenesis and reverse drug resistance of cancer. Recently, it was reported that FASN inhibition induces endoplasmic reticulum stress in cancer cells, and FASN inhibitors cooperate with the endoplasmic reticulum stress inducer to enhance tumour cell death (Little et al, 2007). This suggests that the specificity of FASN inhibitors could be a critical key for successful molecular target therapy of cancer. The expression and activity of Fatty Acid Synthase (FASN; the sole enzyme capable of the reductive de novo synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA, and nicotinamide adenine dinucleotide phosphate -NADPH-) is extremely low in nearly all nonmalignant adult tissues, whereas it is significantly up-regulated or activated in many cancer types, thus creating the potential for a large therapeutic index. Since the pioneering observation that inhibition of FASN activity by the mycotoxin cerulenin preferentially kills cancer cells Well-known FASN inhibitors, the natural product cerulenin have been studied. What is the role of FASN in drug resistance phenomenon. ....................................................................................................................................................................................................................................................................................................................