Abstract
Gemcitabine is a chemotherapeutic that is widely used for the treatment of a variety of haematological malignancies and has become the standard chemotherapy for the treatment of advanced pancreatic cancer. Combinational gemcitabine regimes (e.g.with doxorubicin) are being tested in clinical trials to treat a variety of cancers, including colon cancer. The limited success of these trials has prompted us to pursue a better understanding of gemcitabine's mechanism of cell killing, which could dramatically improve the therapeutic potential of this agent. For comparison, we included gamma irradiation that triggers robust cell cycle arrest and Cr(VI), which is a highly toxic chemical that induces a robust p53-dependent apoptotic response. Gemcitabine induced a potent p53-dependent apoptosis that correlated with the accumulation of pro-apoptotic proteins such as PUMA and Bax. This is accompanied by a drastic reduction in p2l and 14-3-3σ protein levels, thereby significantly sensitizing the cells to apoptosis. In vitro and in vivo studies demonstrated that gemcitabine required PUMA transcription to instigate an apoptotic programme. This was in contrast to Cr(VI)-induced apoptosis that required Bax and was independent of transcription. An examination of clinical colon and pancreatic cancer tissues shows higher p53, p21, 14-3-3σ and Bax expression compared with matched normal tissues, yet there is a near absence of PUMA protein. This may explain why gemcitabine shows only limited efficacy in the treatment of these cancers. Our results raise the possibility that targeting the Bax-dependent cell death pathway, rather than the PUMA pathway, could result in significantly improved patient outcome and prognosis for these cancers.
Highlights
The p53-dependent expression of PUMA has been shown as a key determinant of transcription-dependent apoptosis as the phenotype of [PUMA À / À ] HCT116 cells are strikingly similar to that of [p53 À / À ] HCT116 cells.[8,9] There is a transcription-independent p53 response to cellular stress,[10,11,12] whereas p53-dependent apoptosis can be readily observed in the absence of PUMA correlating with the cytosolic accumulation of p53 and activation of Bax.[13,14] p53 can directly interact with Bax, mediating the latter’s oligomerization inducing the permeabilization of mitochondria
We show that gemcitabine and chromium exposure each instigates p53-dependent apoptosis in contrast to g-IRinduced DNA damage that mediates a potent p53-regulated cell cycle arrest response
Cells exposed to g-IR show elevated levels of expression of the pro-apoptotic genes (Bax, PUMA, Bid), suggesting that expression of these is a default response to DNA damage
Summary
The p53-dependent expression of PUMA has been shown as a key determinant of transcription-dependent apoptosis as the phenotype of [PUMA À / À ] HCT116 cells are strikingly similar to that of [p53 À / À ] HCT116 cells.[8,9] There is a transcription-independent p53 response to cellular stress,[10,11,12] whereas p53-dependent apoptosis can be readily observed in the absence of PUMA correlating with the cytosolic accumulation of p53 and activation of Bax.[13,14] p53 can directly interact with Bax, mediating the latter’s oligomerization inducing the permeabilization of mitochondria. 14-3-3s prior to the induction of apoptosis, whereas the loss of either prior to treatment significantly increased sensitivity to each compound. These compounds were effective in reducing tumour burden of [p53 þ / þ ] HCT116 tumours in mice, they showed differential effects on [PUMA À / À ] and [Bax À / À ] HCT116 tumours that mirrored those observed in vitro. As gemcitabine is currently being used to treat pancreatic and colon cancer with limited efficacy, our results raise the possibility that targeting the Baxdependent apoptotic pathway could offer significantly improved patient outcome
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