Inherent and acquired resistance to paclitaxel in hepatocellular carcinoma: molecular events involved

Abstract

Hepatocellular carcinoma (HCC) is a primary malignancy of the liver and is a major cause of cancer related deaths worldwide. Only 10 to 20% of HCC can be surgically excised. Therefore, chemotherapeutic intervention and treatment is essential for achieving favorable prognosis. However, therapeutic outcome of chemotherapy is generally poor owing to inherent resistance of cancer cells to the treatment or due to development of acquired resistance. To differentiate and delineate the molecular events, we developed drug resistant Hep3B cells (DRC) by treating cells with the increasing concentration of paclitaxel. We also developed a unique single cell clone of Hep3B cells (SCC) by selecting single cell colonies and screening them for resistant phenotype. Interestingly, both DRC and SCC were resistant to paclitaxel in comparison to parental Hep3B cells. We analyzed the contributory factors that may be involved in the development of resistance. As expected, level of P-glycoprotein (P-gp) was elevated in DRC. In addition, Caveolin-1 (Cav-1), Fatty acid synthase (FASN) and Cytochrome P450 (CYP450) protein levels were elevated in DRC whereas in SCC, FASN and CYP450 levels were elevated. Downregulation of these molecules by respective siRNAs and/or by specific pharmacological inhibitors resensitized cells to paclitaxel. Interestingly, these drug resistant cells were also less sensitive to vinblastine, doxorubicin and methotrexate with the exception of cisplatin. Our results suggested that differential levels of P-gp, Cav-1 and FASN play a major role in acquired resistant phenotype whereas FASN level was associated with the presentation of inherent resistant phenotype in HCC.

Figure 1. Selection and characterization of human hepatoma cell line Hep3B cells, DRC and SCC.

(A) 2×10³ cells were plated in 96 well plates and medium was removed at indicated time interval and cell survival was evaluated by MTT assay. (B) Hep3B cells, DRC and SCC (5×10⁵) cells were plated in 35 mm petri plates and allowed to incubate for 24 h. Thereafter cell were processed for FACS analysis. (C) 8×10³ cells were plated in 96 well plates containing 100 µl medium. After 24 h incubation, cells were treated with varying concentrations of paclitaxel for 48 h and cell survival was evaluated by MTT assay. (D) Hep3B cells, DRC and SCC were treated with 1 µM paclitaxel for 48 h and their morphology was observed under vertical microscope and photographed.

Figure 2. Selection and characterization of paclitaxel resistant DRC and SCC clone.

(A) Hep3B cells, DRC and SCC treated with or without 1 µM paclitaxel for 48 h and PARP cleavage was analyzed by immunoblot. (B) 3×10⁴ cells were plated in 12 well plates for 24 h followed by treatment with 5.2 µM paclitaxel for 48 h. Medium was replaced with drug free medium and incubated for additional 18–20 days. Cells were then fixed and stained with crystal violet. (C) 1×10⁵ cells were plated in 12 well plates. After 24 h, ∼500 nCi/well of ³H paclitaxel was added to each well for 12 h and 24 h, respectively. Cells were lysed by adding SDS to respective plates. Counts were taken on Packard. (D) Hep3B cells, DRC and SCC (10^6/ml cells) were loaded with Rhodamine-123 (2 µM) for 30 min at 37°C and efflux at respective time interval was measured by MOFLO in the presence or absence of verapamil.

Figure 3. Western blot analyses of whole cell lysates of Hep3B cells, DRC and SCC showing basal level expression of various molecules.

Representative western blot of basal level expression for various molecules was performed. Briefly, cells were harvested and whole cell lysates were prepared. Proteins were resolved using 8% or 10% SDS-PAGE and then processed for western blotting analysis.

Figure 4. Involvement of P-gp or CYP450 in cancer drug resistance.

(A) Cells were treated with varying concentration of verapamil for 24 h. Thereafter medium was replaced with fresh medium and incubated for additional 48 h. (B) Hep3B cells, DRC and SCC were pretreated with verapamil for 24 and then fresh medium was added with or without paclitaxel. After 48 h treatment, drug containing medium was removed and MTT assay was performed. (C) DRC cells were transfected with control or CYPOR siRNA as per manufacture instruction. After 24 h transfection, cell lysate was prepared and protein was resolved in 10% SDS-PAGE followed by western blotting was performed. (D) Hep3B cells, DRC and SCC were transfected with control or CYPOR siRNA. Twenty-four hours posttransfection, cells were washed and medium was replaced with fresh one with or without paclitaxel for 48 h and cell survival was evaluated by MTT assay.

Figure 5. FASN knockdown sensitizes both acquired and inherent drug resistant cells.

(A) Cells were treated with varying concentration of cerulenin for 24 h. Cells were washed and fresh medium was added for additional 48 h. MTT assay was performed and reading was taken at 570 nm using ELISA plate reader. (B) Hep3B cells, DRC and SCC were pretreated with 30 µM cerulenin. After 24 h treatment, medium was replaced with fresh medium with or without paclitaxel for 48 h and cell survival was evaluated by MTT assay. (C) DRC transfected with control or FASN siRNA for 24 h and western blotting was performed. (D) Hep3B cells, DRC and SCC were transfected with control and FASN siRNA as per manufacture instruction. Twenty-four hours posttransfection, medium with or without paclitaxel was added for 48 h and cell survival was evaluated by MTT assay.

Figure 6. Cav-1 knockdown sensitizes acquired drug resistant clone (DRC) towards paclitaxel.

(A) Hep3B cells, DRC and SCC were treated with MCD for 4 h. Thereafter, fresh medium was added for additional 48 h and cell survival was evaluated by MTT assay. (B) Hep3B cells, DRC and SCC were treated with MCD for 4 h and then fresh medium with or without 300 nM paclitaxel were added for further 48 h. After indicated treatment with or without paclitaxel, medium was removed and MTT assay was performed. (C) DRC cells were transfected with control or Cav-1 siRNA, respectively as per manufacturer instruction. After 36 h, cell lysates were prepared and Cav-1 expression was analyzed by western blotting. (D) 8x10³ cells were plated in 96 well plates and allowed to incubate for 24 h. Cells were transfected with control and Cav-1 siRNA for 36 h and then fresh medium containing 300 nM paclitaxel was added for additional 48 h. Thereafter cell survival was evaluated by MTT assay.

Figure 7. DRC and SCC exhibit cross resistance to different classes of anti-cancer agents.

(A–E) Hep3B cells, DRC and SCC were plated and treated with different concentration of Paclitaxel, Vinblastine, Methotrexate, Doxorubicin and Cisplatin. After respective drug treatment for 48 h, drug containing medium was replaced and cell survival was evaluated by MTT assay.

Figure 8. Cav-1 knockdown inhibit the expression of FASN and vice versa.

(A) DRC (5×10⁵) were plated in 35 mm petri plate. After 24 h, cells were transfected with siRNAs targeting Cav-1, FASN or CYPOR as per manufacturer instruction. Simultaneously, 40 µM verapamil was added for 24 h. Thirty-six hours posttransfection or 24 h verapamil treatment, cells were harvested and lysates were prepared. Fifty microgram whole cell lysate proteins were resolved on 8% or 10% SDS-PAGE and western blot was performed. (B) Hep3B cells, DRC and SCC were treated with 300 nM paclitaxel for 24 and 48 h respectively. Whole cell lysates were prepared and 30 µg was resolved on 10% SDS-PAGE and western blotting was performed. (C) Co-immunoprecipitation of Cav-1 and FASN in Hep3B cells, DRC and SCC was carried out using FASN specific antibody. Cav-1 and FASN were detected in the immune complex by immunoblotting. IgG heavy chain and GAPDH served as loading control. (D and E) Hep3B cells, DRC and SCC were plated and allowed to adhere for 24 h and cells were pretreated with MCD (4 h) or cerulenin (24 h). After inhibitor treatment, paclitaxel was added for additional 48 h. Cells were washed with PBS, fresh medium was added and cells allowed to form colonies for ∼ 21 days. Colonies were stained with crystal violet and photographed.

Figure 9. Schematic representation of proposed mechanism of drug resistance in DRC and SCC.