Combining Paclitaxel with ABT-263 Has a Synergistic Effect on Paclitaxel Resistant Prostate Cancer Cells

<div><p>We assessed the capability of paclitaxel, one of the taxanes, to induce death in two prostate cancer lines, LNCaP and PC3. Paclitaxel drove an apoptotic pathway in LNCaP, but not in PC3 cells, in response to G2/M arrest. An examination of the levels of anti-apoptotic proteins revealed that Bcl-xl was much higher in PC3 cells than in LNCaP cells and Bcl2 could be detected only in PC3 cells, not in LNCaP cells. Knocking down Bcl-xl enhanced paclitaxel-induced apoptosis in LNCaP cells, while we were unable to knock down Bcl-xl efficiently in PC3 cells. Significantly, a comparison of ABT-263, a specific inhibitor of Bcl2 and Bcl-xl, with ABT-199, a Bcl2 selective inhibitor, disclosed that only ABT-263, not ABT-199, could induce apoptosis in LNCaP and PC3 cells. The results indicate that Bcl-xl has a protective role against paclitaxel-induced apoptosis in LNCaP and PC3 cells, and its overexpression causes the paclitaxel resistance seen in PC3 cells. Interestingly, combined paclitaxel with ABT-263 to treat LNCaP and PC3 cells demonstrated synergistic apoptosis activation, indicating that ABT-263 could enhance paclitaxel-induced apoptosis in LNCaP cells and overcome Bcl-xl overexpression to trigger paclitaxel-induced apoptosis in PC3 cells. We also observed that the activation of apoptosis in LNCaP cells was more efficient than in PC3 cells in response to paclitaxel plus ABT-263 or to ABT-263 alone, suggesting that the apoptosis pathway in PC3 cells might have further differences from that in LNCaP cells even after Bcl-xl overexpression is accounted for.</p></div

Caspase-dependent apoptotic pathways were seen in LNCaP, but not in PC3 cells, in response to G2/M arrest.

<p>(A) Immunoblot analysis of cell lysates from synchronized LNCaP and PC3 cells treated by paclitaxel over the indicated time courses for the detection of caspase 3 and PARP with its degradation product. (B) Immunoblot analysis of cell lysates from the adhered or detached fractions of LNCaP or PC3 cells after paclitaxel treatment over time courses for the detection of the cleavage form of PARP. Experiments were repeated three times and representative results are shown. PARP (c): the cleavage form of PARP.</p

The expression of BH3-only protein, pro-apoptotic proteins and anti-apoptotic proteins in LNCaP or PC3 cells after treatment with paclitaxel.

<p>(A) The expression of BH3-only protein Bim and pro-apoptotic proteins including both Bak and Bax, in LNCaP and PC3 cells. (B) The expression of anti-apoptotic proteins including Bcl2, Bcl-xl and Mcl-1 in LNCaP or PC3 cells. Immunoblot analysis of cell lysates from LNCaP or PC3 cells treated by paclitaxel over time as indicated. Experiments were repeated three times and representative results are shown. (C) Comparison of Bim, Bcl-xl and Mcl-1 between LNCaP and PC3 cells with/without paclitaxel treatment. Immunoblot analysis of cell lysates from LNCaP or PC3 cells treated for 48 hr. Experiments were repeated three times and representative results are shown. Immunoblot images of Bim, Bcl-xl, Mcl-1 and α-tubulin in LNCaP and PC3 with/without paclitaxel treatment were quantitated by ImageJ. The readouts, defined as arbitrary light units, of Bim, Bcl-xl and Mcl-1 normalized to the readout of α-tubulin are shown on the right side. (**) statistical significance between two readouts.</p

The effect of ABT-199, ABT-263 or ABT-263 in combination with paclitaxel on LNCaP or PC3 cells.

<p>(A) ABT-263, but not ABT-199, could effectively trigger PARP degradation in LNCaP and PC3 cells in a dose-dependent manner, but it could only activate caspase 3 to a detectable level in LNCaP cells. Immunoblot analysis of cell lysates from LNCaP or PC3 cells treated by ABT-199 or ABT-263 alone for 48 hr at various concentrations were assayed as indicated. Caspase 3(a): the activated form of caspase 3. Caspase 7(a): the activation form of caspase 7. Experiments were repeated three times and representative results are shown. The readouts of caspase 3(a) normalized to GAPDH between LNCaP and PC3 cells treated with 5μM of ABT-263 are shown at the bottom. (**)statistical significance between two readouts. (B) The combination of 50 nM of paclitaxel with various concentrations of ABT-263 had a synergistic effect on apoptosis in both LNCaP and PC3 cells. The efficacy of ABT-263 in combination with paclitaxel in LNCaP cells was higher than in PC3 cells. Immunoblot analysis of cell lysates from LNCaP or PC3 cells treated by paclitaxel in combination with ABT-263 or ABT-263 alone for 48 hr at various concentrations were assayed as indicated. Caspase 3(a): the activated form of caspase 3. Caspase 7(a): the activation form of caspase 7. Experiments were repeated three times and representative results are shown. The respective readouts of caspase 3(a) normalized to internal control, α-tubulin or GAPDH in LNCaP or PC3 with LNCaP are shown at the bottom. (**) statistical significance between two readouts.</p

Paclitaxel induced cell cycle arrest at G2/M in LNCaP and PC3 cells.

<p>(A) Flow cytometric analyses of synchronized PC3 or LNCaP cells treated by paclitaxel through the indicated time courses. Analyses were in triplicate and representative histograms are shown. X- and Y-axes represent DNA content and cell numbers, respectively. (B) Immunofluorescence micrograph of microtubules from synchronized PC3 or LNCaP cells treated by paclitaxel over the indicated time courses. Analyses were duplicated and representative immunofluorescence micrographs are shown. α-Tubulin was shown by green color. DNA was shown by red color.</p

The knockdown effect of Bcl-xl or Mcl-1 in LNCaP or PC3 cells.

<p>Bcl-xl and Mcl-1 knockdown caused increased apoptosis as assessed by the activation of caspase 3 and PARP degradation in LNCaP cells, but not in PC3 cells. LNCaP or PC3 cells were transiently transfected with Bim, Bcl-xl or Mcl-1 siRNA or scrambled siRNA for 24 hr. Cells were treated with/without 50 nM of paclitaxel for 48 hr, and then subjected to immunoblot analysis. Caspase 3(a): the active form of caspase 3. PARP(c): the cleavage form of PARP. Bcl-xl(s): the short exposure image of Bcl-xl. Bcl-xl (l): the long exposure image of Bcl-xl. Experiments were repeated three times and representative results are shown. The respective readouts of the immunoblot images of Bcl-xl, Mcl-1 and caspase 3(a) normalized to α-tubulin in LNCaP cells are shown at the bottom. Only knockdown with/without paclitaxel treatment was quantitated. (**)statistical significance between two readouts.</p