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Hep3Bcellsweretreatedwith increasingconcentrationsofPaclitaxel incellculture mediumfor selection of Paclitaxel-resistant cells. After successive Paclitaxel treatments for duration of ~7 weeks, several drug resistant cell 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 following experiments in the present study. In addition, we also developed a 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 these clone 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 significant change in growth and cell cycle distribution (Fig. 1A). To determine the survival capacityofbothPaclitaxel-sensitive Hep3B and Paclitaxel-resistantcells DRC and SCC cellsweretreatedwith1 µMPaclitaxel for48hrs. Paclitaxel-sensitive Hep3B cells showed blebbing and cell rounding with empty spaces visualized surrounded by the cells. This suggested that a large fraction of these cells were undergone in G2/M phase, with a few of these cells undergoing apoptosis. However, 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). Both assays detected a smaller proportion 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 expression of the cleaved Poly(ADP-ribose)polymerase(c-PARP) protein,anprominentmarkerforcaspase-mediated apoptosis, was also examined afterthe cells weretreated with1 µM Paclitaxel for 48hrs.We observed significantly higher levels of cleaved PARP in sensitive Hep3B cells and correspondingly higher levels of un-cleaved PARP in Paclitaxel-resistant DRC and SCC cells (Fig. 1D). Cell viability assay showed thatDRCandSCCcellscouldtoleratemuchhigherconcentrationsofPaclitaxel comparedto Hep3Bcells,withtheirIC50concentrationswas foundtobemorehigher than Hep3B cells (Fig. 1E).
Increased expression of caveolin-1, FASN and Cytochrome P450 in acquired and inherent Paclitaxel-resistant cells
To examine the role of caveolin-1 in mediating Paclitaxel resistance in human liver cancer cells, the expression of caveolin-1, Fatty acid synthase and cytochrome P450 was examined in Hep3B, DRC and SCC cells. We found that a caveolin-1 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 an 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 drug resistance. For example, Hsps 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 an important role in drug resistance phenomenon. Based on this fact we also checked whether heat shock proteins are also involved in drug resistant phenomenon. We found that Hsp70, Hsp40, Hsp 90 and Hsp27 were not change in Hep3B and its derived clones DRC and SCC. These results indicated that acquired Paclitaxel resistance is correlated with the increased exression of cav-1 in DRC. This is may be due to acquired resistance 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. 2B).We investigatedthe mechanism that may contribute to the augmented expression of caveolin-1, Hep3B cells weretreated with cyloheximideto blockproteinsynthesisandthecellswere treatedwithorwithoutPaclitaxelfor different time interval,the protein stability of caveolin-1 was measured by Western blot(Fig. 2C). The result showedthat caveolin-1 proteinis more stablein Paclitaxeltreated cells thanthat of untreatedcells. Moreover, we also compared the mRNA level of caveolin-1 in Paclitaxel-treated and untreated cells by quantitative RT-PCR (Fig. 2D). The result indicated that Paclitaxel treatment increased the mRNA expression of caveolin-1 as well as protein expression contritribute to the up-regulation of caveolin-1 in acquired drug resistance clone DRC cells.
Implication of increased expression of p-gp in acquired drug resistant cells
Cancers resistant to many anticancer drugs (MDR, or MDR1) commonly express a membrane P-glycoprotein. Pgp, first described by Juliano and Ling, presumably reduced the intracellular concentration of natural product anticancer drugs such as doxycycline by playing a role as a drug efflux pump. Human cancers may intrinsically express the MDR1 Pgp (de novo) or do so after exposure to chemotherapy drugs (acquired expression). Hepatocellular carcinoma represents a cancer subset originating from normal tissues that express Pgp constitutively. To examine the role of p-glycoprotein in mediating Paclitaxel resistance in Hep3B and its derived clones DRC and SCC, the expression of p-glycoprotein was investigated. We observed that a p-glycoprotein level was significantly increased in DRC only (Fig. 3A). This increased expression of p-gp might be playing a role in acquired drug resistance phenomenon. To further confirm this hypothesis, we investigated drug uptake assay with labelled paclitaxel and examine the p-gp activity by drug efflux assay using rhodamine-123. Rhodamine-123 is a p-gp substrate used to measure efflux of rhodamine-123 from cell flow cytometry. Hep3B and its derived clone were loaded with rhodamine-123 followed by washing with drug free media to remove extracellular dye. The remaining rhodamine-123 dye in cells was monitored by flow activated cell sorter. The ï¬‚uorescence intensity alteration of Rh-123 reï¬‚ects the change of P-gp transport activity in the membrane. The higher the ï¬‚uorescence intensity of remaining Rh-123 in the cells, the lower the P-gp transport activity.
The downregulation of caveolin-1, p-gp, FASN and cytochrome P450 re-sensitizes Paclitaxel resistance clones
The increase of caveolin-1, p-gp, FASN and cytochrome P450 expression detected in Paclitaxel-resistant cells suggests that caveolin-1 and p-gp may play a critical role in Paclitaxel resistance in DRC and FASN and cytochrome P450 in SCC. Therefore, the effect of Caveolin-1, FASN and cytochrome P450 downregulation onthesensitivityofPaclitaxelwasinvestigated.Since the expression of FASN, cytochrome P450 was upregulated in inherently resistant SCC and acquiredly DRC and caveolin-1 and p-gp expression was upregulated in DRC only (Fig. 2),wehypothesizedthatthedownregulationofthese molecules by siRNA mightre-sensitize Paclitaxel-resistantcellstoPaclitaxel.Tothisend, caveolin-1 wasknockeddownwithspecific siRNAin DRC and parental Hep3B cells respectively (Fig 3A), andthenthe cells weretreated with incresing concentrations of Paclitaxel. The downregulation of caveolin-1 increased the sensitivity ofthesecellstoPaclitaxel,withacquired-resistant DRC cellsshowingabouta 10 - 40 percentfolddecreasein cell survival under 10- 200 nM Paclitaxeltreatment measured by MTT assay (Fig. 3B) but this difference of sensitivity against paclitaxel was not observed in inherently resistant SCC after knock down of caveolin-1 (Fig. 3C). Interestingly, acquired resistant DRC cells showed a much better overall increased sensitivity to Paclitaxel compared to their parental Hep3B cells cells (Fig. 3B and 3C). Similar assays were performed in another inherently 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. Comparedtothoseofcontrol Hep3Bcells,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. Wefirstexaminedtheeffectof MβCD oncaveolin-1 expression andcellviabilityofHep3BandDRCcells.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-resistantcells,wefurtherinvestigatedtheeffectsofcombiningPaclitaxelwith MΒCD on Paclitaxel-resistant DRC. Paclitaxel combined with MβCD were much more effective in inhibiting cell viability compared with either agent given alone. Similartreatment combinations were performedSCC, 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 Paclitaxelre-sensitization,we examined cellular apoptosisinthese cells. PARP, a nuclear proteinthat can be easily cleavedbycaspases,hasbeenwidelyusedasanapoptosismarker.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 asignificantincrease ofthelevels ofcleaved PARP aftertreatment withthe combination of Paclitaxel and MΒCD comparedtotreatment withsingle agent(Fig. 6D). Thisindicatesthatcellularapoptosisisamechanisminvolvedintheincreasedcell 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)
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 AktAlthough 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.....................................................................................................................................................................................................................................................................................................................