Gemcitabine-mediated tumour regression and p53-dependent gene expression: implications for colon and pancreatic cancer therapy

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.

Figures

Figure 1

p53 is required for gemcitabine- and Cr(VI)-mediated cell death. (a) At 72 h post chemical exposure, MTS assays were conducted for each cell line shown. Stable knockdown cell lines were generated using our retrovirus expression system (pSUPER-Retro) and selected and cultured in puromycin (2 μg/ml) for the duration of our studies. Results are mean±S.D.; ***P<0.0001, *P<0.05. Each assay was conducted in triplicate and each set of experiments was repeated three times. (b) Cell lines were treated (10 μM Gemcitabine, 30 μM Cr(VI) or 10 Gy γ-IR) for 48 h and lysates were prepared and subjected to SDS-PAGE. Immunoblotting was carried out using caspase-3, caspase-9, caspase-2, p53 and β-actin antibodies. (c) At 48 h post DNA damage, annexin-V staining was carried out following the manufacturer's guidelines. The percentage of positive cells was determined by FACS analysis (Becton Dickinson, Franklin Lakes, NJ, USA). Results are mean±S.D. n=3

Figure 2

BH3-family transcription and translation is conserved regardless of damage modality. (a) Indicated cell lines were exposed to gemcitabine (10 μM), Cr(VI) (30 μM) or γ-IR (10 Gy) for 48 h. Immunoblotting was carried out for Bcl-2, Bcl-xL, Bid, Bax, PUMA, p53 and β-actin. (b) Cells were treated as indicated and RNA was extracted using Trizol (Invitrogen). cDNA was generated (superscript II, Invitrogen) and qRT-PCR (Sybr Green, Stratagene) was carried out for Bcl-2, Bcl-xL, Bid, Bax, PUMA and GAPDH. Expression was normalized to GAPDH and gene expression (fold change) was calculated using the 2−ΔΔCT formula. Each assay was conducted in quadruplicate, N=3

Figure 3

Differential expression of p21 and 14-3-3σ affects sensitivity to chemotherapeutics. (a) Indicated cell lines were exposed to gemcitabine (10 μM), Cr(VI) (30 μM) or γ-IR (10 Gy) for 48 h. Immunoblotting was carried out for p21, 14-3-3σ and β-actin. (b) Cells were treated as indicated and qRT-PCR was carried out 24 h post damage for p21, 14-3-3σ and GAPDH. Each assay was conducted in quadruplicate and each set of experiments was repeated three times. (c) The viability of cell lines with p21 or 14-3-3σ knocked out or knocked down was measured after 72 h exposure to the indicated DNA-damaging agents. Results are mean±S.D.; ***P<0.0001. Each assay was conducted in triplicate, N=3

Figure 4

Gemcitabine induces cell death in a transcription-dependent manner. (a) Cell lines indicated were mock/pretreated with actinomycin D (1 μg/ml) for 6 h. At 72 h MTS assays were conducted for the indicated DNA-damaging agents in the presence of actinomycin D. N=3. Results are mean±S.D.; ***P<0.0001. (b) Indicated cell lines were mock/pretreated with actinomycin D (1 μg/ml) and exposed to gemcitabine (10 μM), Cr(VI) (30 μM) or γ-IR (10 Gy) for 48 h. Immunoblotting was carried out for p53, p21, 14-3-3σ, Bax, PUMA and β-actin. (c) Indicated cell lines were treated as described in b. Nuclear and cytoplasmic fractions were collected and immunoblotting was carried out for total p53, actin and Lamin A/C

Figure 5

Gemcitabine requires PUMA to induce cell death. (a) Cells with PUMA or Bax knocked out or knocked down were exposed to gemcitabine for 72 h and MTS assays were conducted. Results are mean±S.D.; ***P<0.0001, *P<0.05. Each assay was conducted in triplicate, N=3. (b) MTS dose studies were repeated 24 h post transient transfection (5 μg) of either the PUMA or Bax expression plasmid. N=3. Results are mean±S.D.; ***P<0.0001, NS, not significant

Figure 6

Gemcitabine-dependent tumour regression correlates with PUMA status. (a) Subcutaneous [p53+/+] or [p53−/−] HCT116 tumours were grown in the right flanks of 8-week-old male NOD/SCID mice. Palpable tumours were injected on day 1 and day 4. Tumours were measured thrice weekly and collected 21 days post treatment or when tumours reached over 1200 mm3 volume. Seven animals were used per group. P-values are indicated for each group. (b) Studies, as described in a, were repeated using the matched [PUMA−/−] or [Bax−/−] HCT116 cell lines. N=7. (c) Tumours from a were collected 21 days post treatment and homogenized. Samples were analyzed by immunoblotting for total p53, p21, 14-3-3σ, Bax, PUMA and β-actin

Figure 7

Primary colon and pancreatic tumours display elevated p53 and attenuated PUMA protein expression. (a) H&E staining was conducted following surgical removal of colon tumour and matched normal tissue. Scale bars (low magnification) indicate 100 μm and 40 μm (high magnification). Immunofluorescence staining was carried out from snap-frozen tumour tissue immediately after surgical removal. Sections were stained for ANXA2 (cell surface marker) and p53. (b) Colon (N=8) and pancreatic (N=6) tumour samples (including matched normal tissue) were surgically removed. qRT-PCR was conducted for p21, 14-3-3σ, Bax, PUMA and GAPDH in quadruplicate (c) Colon tumour, pancreatic tumour and matched normal tissue were surgically removed and homogenized, and proteins were extracted. Samples (100 μg) were analyzed by immunoblotting for HIF-1α, Phospho-Ser15 p53, total p53, β-actin, p21 14-3-3σ, PUMA and Bax. (d) The average amount of total p53 protein was quantified and the mean average±S.D. (N=8 and 6, respectively) was plotted for both of our normal and cancer samples. (e) Fifty microgram of the total protein extract from c was incubated with our generated, specific biotinylated promoter sequences for the p21, Bax or PUMA genes addressing p53 recruitment/binding. Following capture, protein/DNA complexes were separated using streptavidin beads (Invitrogen). Proteins were removed, denatured and analysed by immunobotting for total p53. Non-captured/promoter bound p53 protein was similarly analyzed. (f) Each sample (bound/unbound p53) from each sample (normal or colon cancer) was quantified and the percentage of bound p53 protein was calculated. The mean average percentage ±S.D. was plotted and P-values comparing the percentage bound between normal/cancer samples was calculated

Figure 8

Proposed ‘arms' of the p53 apoptotic response. Under apoptotic conditions (e.g. gemcitabine or CrVI)), p53 is activated and accumulates. Following gemcitabine exposure, p53 accumulation is predominately nuclear inducing the transcription of PUMA (in addition to Bax and other p53-regulated genes). Following the accumulation of PUMA protein, there is a significant induction of PUMA-dependent/transcription-dependent apoptosis. In contrast, Cr(VI) exposure induces p53 accumulation (and subsequent transcription of Bax and PUMA); however, this is not a crucial requirement to induce apoptosis as there is a significant accumulation of p53 (and Bax) protein within the cytoplasm of Cr(VI)-exposed cells. As a result, Cr(VI) induces a potent Bax-dependent/transcription-independent apoptotic response characterized by p53 and BAx protein accumulation in the cytoplasm of exposed cells. Regardless of the crucial effector protein, both modalities ablate p21 and 14-3-3σ expression, priming the cell to induce apoptosis