CITED2 silencing sensitizes cancer cells to cisplatin by inhibiting p53 trans-activation and chromatin relaxation on the ERCC1 DNA repair gene

In this study, we show that silencing of CITED2 using small-hairpin RNA (shCITED2) induced DNA damage and reduction of ERCC1 gene expression in HEK293, HeLa and H1299 cells, even in the absence of cisplatin. In contrast, ectopic expression of ERCC1 significantly reduced intrinsic and induced DNA damage levels, and rescued the effects of CITED2 silencing on cell viability. The effects of CITED2 silencing on DNA repair and cell death were associated with p53 activity. Furthermore, CITED2 silencing caused severe elimination of the p300 protein and markers of relaxed chromatin (acetylated H3 and H4, i.e. H3K9Ac and H3K14Ac) in HEK293 cells. Chromatin immunoprecipitation assays further revealed that DNA damage induced binding of p53 along with H3K9Ac or H3K14Ac at the ERCC1 promoter, an effect which was almost entirely abrogated by silencing of CITED2 or p300. Moreover, lentivirus-based CITED2 silencing sensitized HeLa cell line-derived tumor xenografts to cisplatin in immune-deficient mice. These results demonstrate that CITED2/p300 can be recruited by p53 at the promoter of the repair gene ERCC1 in response to cisplatin-induced DNA damage. The CITED2/p300/p53/ERCC1 pathway is thus involved in the cell response to cisplatin and represents a potential target for cancer therapy.     

Golgi-SNARE GS28 potentiates cisplatin-induced apoptosis by forming GS28-MDM2-p53 complexes and by preventing the ubiquitination and degradation of p53

In the present study, we observed that the Golgi-SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) GS28 forms a complex with p53 in HEK (human embryonic kidney)-293 cells. Given that p53 represents a tumour suppressor that affects the sensitivity of cancer cells to various chemotherapeutic drugs, we examined whether GS28 may influence the level of sensitivity to the DNA-damaging drug cisplatin. Indeed, knockdown of GS28 using short-hairpin RNA (shGS28) induced resistance to cisplatin in HEK-293 cells. On the other hand, overexpression of GS28 sensitized HEK-293 cells to cisplatin, whereas no sensitization effect was noted for the mitotic spindle-damaging drugs vincristine and taxol. Accordingly, we observed that knockdown of GS28 reduced the accumulation of p53 and its pro-apoptotic target Bax. Conversely, GS28 overexpression induced the accumulation of p53 and Bax as well as the pro-apoptotic phosphorylation of p53 on Ser(46). Further experiments showed that these cellular responses could be abrogated by the p53 inhibitor PFT-α (pifithrin-α), indicating that GS28 may affect the stability and activity of p53. The modulatory effects of GS28 on cisplatin sensitivity and p53 stability were absent in lung cancer H1299 cells which are p53-null. As expected, ectopic expression of p53 in H1299 cells restored the modulatory effects of GS28 on sensitivity to cisplatin. In addition, GS28 was found to form a complex with the p53 E3 ligase MDM2 (murine double minute 2) in H1299 cells. Furthermore, the ubiquitination of p53 was reduced by overexpression of GS28 in cells, confirming that GS28 enhances the stability of the p53 protein. Taken together, these results suggest that GS28 may potentiate cells to DNA-damage-induced apoptosis by inhibiting the ubiquitination and degradation of p53.     

The regulation of p53, p38 MAPK,JNK and XBP-1s by sphingosine kinases in human embryonic kidney cells

Since inhibitors of sphingosine kinases (SK1, SK2) have been shown to induce p53-mediated cell death, we have further investigated their role in regulating p53, stress activated protein kinases and XBP-1s in HEK293T cells. Treatment of these cells with the sphingosine kinase inhibitor, SKi, which fails to induce apoptosis, promoted the conversion of p53 into two proteins with molecular masses of 63 and 90 kDa, and which was enhanced by over-expression of ubiquitin. The SKi induced conversion of p53 to p63/p90 was also enhanced by siRNA knockdown of SK1, but not SK2 or dihydroceramide desaturase (Degs1), suggesting that SK1 is a negative regulator of this process. In contrast, another sphingosine kinase inhibitor, ABC294640 only very weakly stimulated formation of p63/p90 and induced apoptosis of HEK293T cells. We have previously shown that SKi promotes the polyubiquitination of Degs1, and these forms positively regulate p38 MAPK/JNK pathways to promote HEK293T cell survival/growth. siRNA knockdown of SK1 enhanced the activation of p38 MAPK/JNK pathways in response to SKi, suggesting that SK1 functions to oppose these pro-survival pathways in HEK293T cells. SKi also enhanced the stimulatory effect of the proteasome inhibitor, MG132 on the expression of the pro-survival protein XBP-1s and this was reduced by siRNA knockdown of SK2 and increased by knockdown of p53. These findings suggest that SK1 and SK2 have opposing roles in regulating p53-dependent function in HEK293T cells.     

Inhibition of NAMPT pathway by FK866 activates the function of p53 in HEK293T cells

Inactivation of p53 protein by endogenous and exogenous carcinogens is involved in the pathogenesis of different human malignancies. In cancer associated with SV-40 DNA tumor virus, p53 is considered to be non-functional mainly due to its interaction with the large T-antigen. Using the 293T cell line (HEK293 cells transformed with large T antigen) as a model, we provide evidence that p53 is one of the critical downstream targets involved in FK866-mediated killing of 293T cells. A reduced rate of apoptosis and an increased number of cells in S-phase was accompanied after knockdown of p53 in these cells. Inhibition of NAMPT by FK866, or inhibition of SIRT by nicotinamide decreased proliferation and triggered death of 293T cells involving the p53 acetylation pathway. Additionally, knockdown of p53 attenuated the effect of FK866 on cell proliferation, apoptosis, and cell cycle arrest. The data presented here shed light on two important facts: (1) that p53 in 293T cells is active in the presence of FK866, an inhibitor of NAMPT pathway; (2) the apoptosis induced by FK866 in 293T cells is associated with increased acetylation of p53 at Lys382, which is required for the functional activity of p53.     

A New Isoquinolinium Derivative, Cadein1, Preferentially Induces Apoptosis in p53-defective Cancer Cells with Functional Mismatch Repair via a p38-dependent Pathway

We screened a protoberberine backbone derivative library for compounds with anti-proliferative effects on p53-defective cancer cells. A compound identified from this small molecule library, cadein1 (cancer-selective death inducer 1), an isoquinolinium derivative, effectively leads to a G₂/M delay and caspase-dependent apoptosis in various carcinoma cells with non- functional p53. The ability of cadein1 to induce apoptosis in p53-defective colon cancer cells was tightly linked to the presence of a functional DNA mismatch repair (MMR) system, which is an important determinant in chemosensitivity. Cadein1 was very effective in MMR⁺/p53⁻ cells, whereas it was not effective in p53⁺ cells regardless of the MMR status. Consistently, when the function of MMR was blocked with short hairpin RNA in SW620 (MMR⁺/p53⁻) cells, cadein1 was no longer effective in inducing apoptosis. Besides, the inhibition of p53 increased the pro-apoptotic effect of cadein1 in HEK293 (MMR⁺/p53⁺) cells, whereas it did not affect the response to cadein1 in RKO (MMR⁻/p53⁺) cells. The apoptotic effects of cadein1 depended on the activation of p38 but not on the activation of Chk2 or other stress-activated kinases in p53-defective cells. Taken together, our results show that cadein1 may have a potential to be an anti-cancer chemotherapeutic agent that is preferentially effective on p53-mutant colon cancer cells with functional MMR.     

Cisplatin-induced non-apoptotic death of pancreatic cancer cells requires mitochondrial cyclophilin-D-p53 signaling

The pancreatic cancer remains a fatal disease for the majority of patients. Cisplatin has displayed significant cytotoxic effects against the pancreatic cancer cells, however the underlying mechanisms remain inconclusive. Here, we found that cisplatin mainly induced non-apoptotic death of the pancreatic cancer cells (AsPC-1 and Capan-2), which was associated with a significant p53 activation (phosphorylation and accumulation). Further, activated p53 was found to translocate to mitochondria where it formed a complex with cyclophilin D (Cyp-D). We provided evidences to support that mitochondrial Cyp-D/p53 complexation might be critical for cisplatin-induced non-apoptotic death of pancreatic cancer cells. Inhibition of Cyp-D by its inhibitor cyclosporine A (CsA), or by shRNA-mediated knockdown suppressed cisplatin-induced pancreatic cancer cell death. Both CsA and Cyp-D knockdown also disrupted the Cyp-D/p53 complex formation in mitochondria. Meanwhile, the pancreatic cancer cells with p53 knockdown were resistant to cisplatin. On the other hand, HEK-293 over-expressing Cyp-D were hyper-sensitive to cisplatin. Interestingly, camptothecin (CMT)-induced pancreatic cancer cell apoptotic death was not affected CsA or Cyp-D knockdown. Together, these data suggested that cisplatin-induced non-apoptotic death requires mitochondria Cyp-D-p53 signaling in pancreatic cancer cells.     

Δ133p53 decreases the chemosensitivity of carcinoma cell line H1299

The research evaluated the effect of Δ133p53 on the chemosensitivity of lung adenocarcinoma cell line H1299. By this study, the drug‐resistant molecular marker and a new target for cancer therapy could be provided. Δ133p53 or negative control plasmid were transferred into H1299 cells by lentivirus vector. The expression of Δ133p53 in transfected cells was examined using immunofluorescence. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) method and colony formation test were applied to detect drug sensitivity after cisplatin or 5‐fluorouracil (5‐FU) treatment. After cisplatin (CDDP)/FU treatment, MTT assay demonstrated that the inhibition rate of H1299/Δ133p53 cell was reduced compared with that of the H1299 and H1299/NEG cells at the same concentration of drug. The 50% inhibitory concentrations (IC ₅₀) of CDDP and 5‐FU rose by 36.1 and 30.2%, respectively (P < 0.05). The colony formation assay suggested that the cell proliferation ability of H1299/Δ133p53 cell was prominently increased when compared with that of control group H1299 and H1299 /NEG cells (P < 0.05). The present study demonstrated that the transfection of the Δ133p53 gene in H1299 cells led to the reduction of chemosensitivity.     

Exosomes Transfer p53 between Cells and Can Suppress Growth and Proliferation of p53-Negative Cells

Exosomes are nanosized vesicles that are secreted by many types of cells. We have found that exosomes secreted by HEK293 and HT-1080 can suppress growth and proliferation of p53-deficient cells. Upon overexpression of exogenous p53-GFP in HEK293 cells, we observed p53 protein in exosomes that were secreted by these cells. We also found endogenous p53 in exosomes that were secreted by HT-1080 cells with a higher level of p53 expression. We were able to detect endogenous p53 protein in exosomes that originated from human plasma and were transferred to p53-deficient cells. Our findings indicate that p53 protein can be transferred between cells and may play an important physiological role.     

NSC109268 potentiates cisplatin-induced cell death in a p53-independent manner

Background

Ovarian cancer is the leading cause of death among gynecological cancers. Cisplatin is one of the most effective anticancer drugs used in the treatment of ovarian cancer. Development of resistance to cisplatin limits its therapeutic use. Most of the anticancer drugs, including cisplatin, are believed to kill cancer cells by inducing apoptosis and a defect in apoptotic signaling can contribute to drug resistance. The tumor suppressor protein p53 plays a critical role in DNA damage-induced apoptosis. During a yeast-based drug screening, NSC109268 was identified to enhance cellular sensitivity to cisplatin. The objective of the present study is to determine if p53 is responsible for cisplatin sensitization by NSC109268.

Results

NSC109268 enhanced sensitivity of ovarian cancer 2008 cells and its cisplatin resistant counterpart 2008/C13* cells which express wild-type p53. The potentiation of cisplatin sensitivity by NSC109268 was greater in 2008/C13* cells compared to 2008 cells. Cisplatin caused a concentration-dependent increase in p53 in 2008 and 2008/C13* cells, and the induction of p53 correlated with cisplatin-induced apoptosis as determined by the cleavage of PARP. NSC109268 alone had no effect on p53 but it enhanced p53 level in response to cisplatin. Knockdown of p53 by siRNA, however, did not attenuate cell death in response to cisplatin or combination of NSC109268 and cisplatin.

Conclusions

These results demonstrate that NSC109268 enhances sensitivity of ovarian cancer 2008 cells to cisplatin independent of p53.     

Promoter methylation of O(6)-methylguanine-DNA-methyltransferase in lung cancer is regulated by p53

Methylation of the O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with G:C to A:T transitions in the p53 gene in various human cancers, including lung cancer. In tumors with p53 mutation, MGMT promoter methylation is more common in advanced tumors than in early tumors. However, in tumors with wild-type p53, MGMT promoter methylation is independent of tumor stage. To elucidate whether p53 participates in MGMT promoter methylation, we engineered three cell models: A549 cells with RNA interference (RNAi)-mediated knockdown of p53, and p53 null H1299 cells transfected with either wild-type p53 (WT-p53) or mutant-p53 (L194R, and R249S-p53). Knockdown of endogenous p53 increased MGMT promoter methylation in A549 cells, and transient expression of WT-p53 in p53 null H1299 cells diminished MGMT promoter methylation, whereas the MGMT promoter methylation status were unchanged by expression of mutant-p53. Previous work showed that p53 modulates DNA-methyltransferase 1 (DNMT1) expression; we additionally examined chromatin remodeling proteins expression levels of histone deacetylase 1 (HDAC1). We found that p53 knockdown elevated expression of both DNMT1 and HDAC1 in A549 cells. Conversely, expressing WT-p53 in p53 null H1299 cells reduced DNMT1 and HDAC1 expression, but the reduction of both proteins was not observed in expressing mutant-p53 H1299 cells. CHIP analysis further showed that DNMT1 and HDAC1 binding to the MGMT promoter was increased by MGMT promoter methylation and decreased by MGMT promoter demethylation. In conclusion, MGMT promoter methylation modulated by p53 status could partially promote p53 mutation occurrence in advanced lung tumors.     

X-ray irradiation altered chemosensitivity of a p53-null non-small cell lung cancer cell line

Radiotherapy is an effective approach to treating many types of cancer. Recent progress in radiotherapy technology, such as intensity-modulated radiation therapy (IMRT) and three-dimensional (3D) radiotherapy, allow precise energy transfer to the tumor, which has improved local control rates. However, the emergence of tolerant cells during or after radiotherapy remains problematic. In the present study, we first established a cell population from H1299, the p53-null non-small cell lung cancer cell line, by 10 Gy irradiation using 6 MV X-rays. The radio- and chemosensitivity of this cell population (referred to as H1299-IR) was determined using colony formation analyses and MTS assays. Compared with the parental cell line, the radiosensitivity of H1299-IR was apparently the same. H1299 and H1299-IR were both more radio tolerant than the A549 cell line. However, H1299-IR became significantly more sensitive to cisplatin, an antitumor agent. After exposure to 25 mug/ml cisplatin for 2 h, parental cells steadily grew during the MTS assay, whereas the sensitivity of H1299-IR cells doubled both at 24 and 48 h. Microarray analysis of over 30,000 H1299-IR genes (Agilent Technology) revealed that 12 and 15 genes were up- (> 2.0) and down- (< 2.0) regulated, respectively. Rad51d (homologous recombination repair protein) gene was down-regulated 2.8-fold, whereas matrix metalloproteinase 1 (collagenase-1) gene was up-regulated 4.4-fold. These results indicated that some p53-null non-small cell lung cancers could be successfully treated when X-ray radiotherapy was administered with subsequent or concurrent cisplatin chemotherapy.     

Inhibition of AMP-activated protein kinase sensitizes cancer cells to cisplatin-induced apoptosis via hyper-induction of p53

Cisplatin is one of the most effective and widely used chemotherapeutic agents. However, one of the most salient limitations to the clinical application of cisplatin is the acquired or intrinsic drug resistance exhibited by some tumors. In the present study, we have assessed the potential of an intracellular energy balancing system as a target for augmentation of cisplatin sensitivity in tumors. AMP-activated protein kinase (AMPK) regulates the energy balance system by monitoring intracellular energy status. Here we demonstrate that AMPK is rapidly activated by cisplatin in AGS and HCT116 cancer cells. The inhibition of AMPK in those cells and in xenografts of HCT116 resulted in a remarkable increase in cisplatin-induced apoptosis, which was associated with hyper-induction of the tumor suppressor p53. We further showed that ERK, but not ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) kinases, was involved in the hyper-induction of p53 by the inhibition of cisplatin-induced AMPK. By way of contrast, cisplatin did not induce AMPK activation in HeLa cells, which appear to have a relatively high sensitivity to cisplatin-induced cytotoxicity, but expression of the constitutive active form of AMPK in HeLa cells resulted in a significant increase of cell viability after cisplatin treatment. Collectively, our data suggest that AMPK performs a pivotal function for protection against the cytotoxic effect of cisplatin, thereby implying that AMPK is one of the cellular factors determining the cellular sensitivity to cisplatin. On the basis of these observations, we propose that a strategy combining cisplatin and AMPK inhibition could be developed into a novel chemotherapeutic modality.