Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.     

p53 induces miR-199a-3p to suppress mechanistic target of rapamycin activation in cisplatin-induced acute kidney injury

How p53 participates in acute kidney injury (AKI) progress and what are the underlying mechanisms remain illusive. For this issue, it is important to probe into the role of p53 in cisplatin-induced AKI. We find that p53 was upregulated in cisplatin-induced AKI, yet, pifithrin-α inhibites the p53 expression to attenuated renal injury and cell apoptosis both in vivo cisplatin-induced AKI mice and in vitro HK-2 human renal tubular epithelial cells. To knock down p53 by siRNA significantly decreased the miRNA, miR-199a-3p, expression in HK-2 cells. Blockade of miR-199a-3p significantly reduced cisplatin-induced cell apoptosis and inhibited caspase-3 activity. Mechanistically, we identified that miR-199a-3p directly bound to mechanistic target of rapamycin (mTOR) 3′-untranslated region and overexpressed miR-199a-3p reduce the expression and phosphorylation of mTOR. Furthermore, we demonstrated that p53 inhibited mTOR activation through activating miR-199a-3p. In conclusion, our findings reveal that p53, upregulating the expression of miR-199a-3p affects the progress of cisplatin-induced AKI, which might provide a promising therapeutic target of AKI.     

Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis

Caspases and c-Jun N-terminal kinase (JNK) are activated in tumor cells during induction of apoptosis. We investigated the signaling cascade and function of these enzymes in cisplatin-induced apoptosis. Treatment of Jurkat T-cells with cisplatin induced cell death with DNA fragmentation and activation of caspase and JNK. Bcl-2 overexpression suppressed activation of both enzymes, whereas p35 and CrmA inhibited only the DEVDase (caspase-3-like) activity, indicating that the activation of these enzymes may be differentially regulated. Cisplatin induced apoptosis with the cytochrome c release and caspase-3 activation in both wild-type and caspase-8-deficient JB-6 cells, while the Fas antibody induced these apoptotic events only in wild-type cells. This indicates that caspase-8 activation is required for Fas-mediated apoptosis, but not cisplatin-induced cell death. On the other hand, cisplatin induced the JNK activation in both the wild-type and JB-6 cells, and the caspase-3 inhibitor Z-DEVD-fmk did not inhibit this activation. The JNK overexpression resulted in a higher JNK activity, AP-1 DNA binding activity, and metallothionein expression than the empty vector-transfected cells following cisplatin treatment. It also partially protected the cells from cisplatin-induced apoptosis by decreasing DEVDase activity. These data suggest that the cisplatin-induced apoptotic signal is initiated by the caspase-8-independent cytochrome c release, and the JNK activation protects cells from cisplatin-induced apoptosis via the metallothionein expression.     

Acquisition of chemoresistance in intrahepatic cholangiocarcinoma cells by activation of AKT and extracellular signal-regulated kinase (ERK)1/2

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignant tumor and is refractory to conventional chemotherapy. The aim of this study is therefore to elucidate the mechanism of chemoresistance in ICC which is not fully understood. We generated cisplatin resistant ICC cells via long term exposure to cisplatin and found that these cells are also resistant to 5-fluorouracil (5-FU) and gemcitabine. The chemoresistant cells showed enhanced Bcl-2 expression and reduced Bax expression compared to parental ICC cells. In addition, the resistant cells showed enhanced activation of AKT and extracellular signal-regulated kinase (ERK) 1/2. Inhibition of AKT activation by phosphoinocitide 3-kinase (PI3K) inhibitor LY294002 resulted in reduced Bcl-2 expression and enhanced Bax expression and thus induced apoptosis in the resistant cells, whereas inhibition of ERK1/2 activation by mitogen-activated protein kinase (MEK) inhibitor U0126 did not induce apoptosis without affecting the expression of Bcl-2 and Bax but decreased cell growth. Moreover, the inhibition of AKT or ERK1/2 sensitized the resistant cells to cisplatin and therefore resulted in greatly enhanced cisplatin-induced apoptosis and growth inhibition in the cells. The results indicate that AKT and ERK1/2 signaling mediate chemoresistance in the cells and could be important therapeutic targets for overcoming chemoresistance in ICC.     

SIRT1 activation by resveratrol ameliorates cisplatin-induced renal injury through deacetylation of p53

Nephrotoxicity is one of the important dose-limiting factors during cisplatin treatment. There is a growing body of evidence that activation of p53 has a critical role in cisplatin-induced renal apoptotic injury. The nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 decreases apoptosis through deacetylating of p53, and resveratrol is known as an activator of SIRT1. To study the role of SIRT1 in cisplatin-induced renal injury through interaction with p53, mouse proximal tubular cells (MPT) were treated with cisplatin and examined the expression level of SIRT1, acetylation of p53, PUMA-α, Bax, the cytosolic/mitochondrial cytochrome c ratio, and active caspase-3. The expression of SIRT1 was decreased by cisplatin. Resveratrol, a SIRT1 activator, ameliorated cisplatin-induced acetylation of p53, apoptosis, and cytotoxicity in MPT cells. In addition, resveratrol remarkably blocked cisplatin-induced decrease of Bcl-xL in MPT cells. Further specific SIRT1 inhibition with EX 527 or small interference RNA specific to SIRT1 reversed the effect of resveratrol on cisplatin-induced toxicity. Inhibition of p53 by pifithrin-α reversed the effect of EX527 in protein expression of PUMA-α, Bcl-xL, and caspase-3 and cytotoxicity in MPT cells. SIRT1 protein expression after cisplatin treatment was significantly decreased in the kidney. SIRT1 activation by resveratrol decreased cisplatin-induced apoptosis while improving the glomerular filtration rate. Taken together, our findings suggest that the modulation of p53 by SIRT1 could be a possible target to attenuate cisplatin-induced kidney injury.     

Cisplatin-induced cell death in Saccharomyces cerevisiae is programmed and rescued by proteasome inhibition

Cisplatin is a highly effective chemotherapeutic drug used in the treatment of several tumors. It is a DNA-damaging agent that induces apoptosis of rapidly proliferating cells, an important factor underlying its therapeutic efficacy. Unfortunately, cellular resistance occurs often. A large fraction of tumor cells harbor mutations in p53, contributing to defects in apoptotic pathways and drug resistance. However, cisplatin-induced apoptosis can also occur in p53 deficient cells; thus, elucidation of the molecular mechanism involved will potentially yield new strategies to eliminate tumors that have defects in the p53 pathway. Most of the studies in this field have been conducted in cultured mammalian cells, not amenable to systematic genetic manipulation. Therefore, we aimed to establish a simplified model devoid of a p53 ortholog to study cisplatin-induced programmed cell death (PCD), using the yeast Saccharomyces cerevisiae.Our results indicate cisplatin induces an active form of cell death in yeast, as this process was partially dependent on de novo protein synthesis and did not lead to loss of membrane integrity. Cisplatin also increased DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction other than partial fragmentation. Co-incubation with the proteasome inhibitor MG132 increased resistance to cisplatin and, accordingly, yeast strains deficient in proteasome activity were more resistant to cisplatin than wild-type strains. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Our results indicate inhibition of the proteasome protects budding yeast from cisplatin-induced cell death and validate yeast as a model to study the role of the proteasome in cisplatin-induced PCD. Elucidation of this mechanism will aid in the development of new strategies to increase the efficacy of chemotherapy.     

Integrins regulate microtubule nucleating activity of centrosome through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling

Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and γ-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin β1 tail and show that this mutant substantially decreases microtubule nucleation and γ-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of γ-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.     

Fragmented mitochondria are sensitized to Bax insertion and activation during apoptosis

Recent studies have shown mitochondrial fragmentation during cell stress and have suggested a role for the morphological change in mitochondrial injury and ensuing apoptosis. However, the underlying mechanism remains elusive. Here we demonstrate that mitochondrial fragmentation facilitates Bax insertion and activation in mitochondria, resulting in the release of apoptogenic factors. In HeLa cells, overexpression of mitofusins attenuated mitochondrial fragmentation during cisplatin- and azide-induced cell injury, which was accompanied by less apoptosis and less cytochrome c release from mitochondria. Similar effects were shown by inhibiting the mitochondrial fission protein Drp1 with a dominant negative mutant (dn-Drp1). Mitofusins and dn-Drp1 did not seem to significantly affect Bax translocation/accumulation to mitochondria; however, they blocked Bax insertion and activation in mitochondrial membrane. Consistently, in rat kidney proximal tubular cells, small interfering RNA knockdown of Drp1 prevented mitochondrial fragmentation during azide-induced ATP depletion, which was accompanied by less Bax activation, insertion, and oligomerization in mitochondria. These cells released less cytochrome c and AIF from mitochondria and showed significantly lower apoptosis. Finally, mitofusin-null mouse embryonic fibroblasts (MEF) had fragmented mitochondria. These MEFs were more sensitive to cisplatin-induced Bax activation, release of cytochrome c, and apoptosis. Together, this study provides further support for a role of mitochondrial fragmentation in mitochondrial injury and apoptosis. Mechanistically, mitochondrial fragmentation may sensitize the cells to Bax insertion and activation in mitochondria, facilitating the release of apoptogenic factors and consequent apoptosis.     

Phosphoinositide 3-OH kinase (PI3K) and PKB/Akt delay the onset of p53-mediated, transcriptionally dependent apoptosis.

The phosphoinositide 3-OH kinase (PI3K)-PKB/Akt signaling pathway has been shown to mediate both Ras- and cytokine-induced protection from apoptosis. In addition, apoptosis induced by the p53 tumor suppressor protein can be inhibited by Ras- and cytokine-mediated signaling pathways. It was therefore of interest to determine if the PI3K-PKB/Akt signaling pathway was capable of conferring protection from apoptosis induced by p53. We demonstrate in this report that constitutively active PI3K and PKB/Akt are capable of significantly delaying the onset of p53-mediated apoptosis. This was manifested as a delay in the kinetics of DNA degradation and cell death as well as a profound attenuation in the accumulation of cells with a sub-G(1) DNA content. Moreover, we found that this effect is mediated in the absence of changes in expression of Bcl-2, Bcl-Xl, and the pro-apoptotic protein Bax. Our results provide the first direct and unambiguous link between p53-mediated apoptosis and the PI3K-PKB/Akt signaling pathway.     

Cordycepin inhibits migration of human glioblastoma cells by affecting lysosomal degradation and protein phosphatase activation

Cordycepin, a nucleoside-derivative-isolated form Cordyceps militaris, has been reported to suppress tumor cell proliferation and cause apoptosis. This study investigates the effect of cordycepin on the migration of human glioblastoma cells. Cordycepin suppressed the migration of the human glioblastoma cell lines U87MG and LN229 in transwell and wound healing assays. Cordycepin decreased protein expression of integrin α1, focal adhesion kinase (FAK), p-FAK, paxillin and p-paxillin. The lysosomal inhibitor NH₄Cl blocked the ability of cordycepin to inhibit focal adhesion protein expression and glioma cell migration. In addition, the protein phosphatase inhibitors calyculin A and okadaic acid blocked the cordycepin-mediated reduction in p-Akt, p-FAK and migration. Hematoxylin and eosin staining of mouse xenografts demonstrated that cordycepin reduced brain tumor size in vivo. In conclusion, cordycepin inhibited migration of human glioblastoma cells by affecting lysosomal degradation and protein phosphatase activation. This pathway may be a useful target for clinical therapy in the future.     

Depletion of endogenous nitric oxide enhances cisplatin-induced apoptosis in a p53-dependent manner in melanoma cell lines

The expression of inducible nitric-oxide synthase in melanoma tumor cells was recently shown to correlate strongly with poor patient survival after combination biochemotherapy (p<0.001). Furthermore, evidence suggests that nitric oxide, a reaction product of nitric oxide synthase, exhibits antiapoptotic activity in melanoma cells. We therefore hypothesized that nitric oxide antagonizes chemotherapy-induced apoptosis. Whether nitric oxide is capable of regulating cell growth and apoptotic responses to cisplatin treatment in melanoma cell lines was evaluated. We demonstrate herein that depletion of endogenously produced nitric oxide can inhibit melanoma proliferation and promote apoptosis. Moreover, our data indicate that the depletion of nitric oxide leads to changes in cell cycle regulation and enhances cisplatin-induced apoptosis in melanoma cells. Strikingly, we observed that the depletion of nitric oxide inhibits cisplatin-induced wild type p53 accumulation and p21(Waf1/Cip1/Sdi1) expression in melanoma cells. When cisplatin-induced p53 binding to the p21(Waf1/Cip1/Sdi1) promoter was examined, it was found that nitric oxide depletion significantly reduced the presence of p53-DNA complexes after cisplatin treatment. Furthermore, dominant negative inhibition of p53 activity enhanced cisplatin-induced apoptosis. Together, these data strongly suggest that endogenously produced nitric oxide is required for cisplatin-induced p53 activation and p21(Waf1/Cip1/Sdi1) expression, which can regulate melanoma sensitivity to cisplatin.     

Farnesoid X Receptor Ligand Prevents Cisplatin-Induced Kidney Injury by Enhancing Small Heterodimer Partner

The farnesoid X receptor (FXR) is mainly expressed in liver, intestine and kidney. We investigated whether 6-ethyl chenodeoxycholic acid (6ECDCA), a semisynthetic derivative of chenodeoxycholic aicd (CDCA, an FXR ligand), protects against kidney injury and modulates small heterodimer partner (SHP) in cisplatin-induced kidney injury. Cisplatin inhibited SHP protein expression in the kidney of cisplatin-treated mice and human proximal tubular (HK2) cells; this effect was counteracted by FXR ligand. Hematoxylin and eosin staining revealed the presence of tubular casts, obstructions and dilatations in cisplatin-induced kidney injury, which was attenuated by FXR ligand. FXR ligand also attenuated protein expression of transforming growth factor-β1 (TGF-β1), Smad signaling, and the epithelial-to-mesenchymal transition process, inflammatory markers and cytokines, and apoptotic markers in cisplatin-treated mice. Cisplatin induced NF-κB activation in HK2 cell; this effect was attenuated by pretreatment with FXR ligand. In SHP knockdown by small interfering RNA, cisplatin-induced activation of TGF-β1, p-JNK and Bax/Bcl-2 ratio was not attenuated, while SHP overexpression and FXR ligand inhibited expression of these proteins in cisplatin-pretreated HK2 cells. In conclusion, FXR ligand, 6ECDCA prevents cisplatin-induced kidney injury, the underlying mechanism of which may be associated with anti-fibrotic, anti-inflammatory, and anti-apoptotic effects through SHP induction.     

Protein Kinase Cα (PKCα) Regulates p53 Localization and Melanoma Cell Survival Downstream of Integrin αv in Three-dimensional Collagen and in Vivo

Protein kinase C α (PKCα) is overexpressed in numerous types of cancer. Importantly, PKCα has been linked to metastasis of malignant melanoma in patients. However, it has been unclear how PKCα may be regulated and how it exerts its role in melanoma. Here, we identified a role for PKCα in melanoma cell survival in a three-dimensional collagen model mimicking the in vivo pathophysiology of the dermis. A pathway was identified that involved integrin αv-mediated up-regulation of PKCα and PKCα-dependent regulation of p53 localization, which was connected to melanoma cell survival. Melanoma survival and growth in three-dimensional microenvironments requires the expression of integrin αv, which acts to suppress p53 activity. Interestingly, microarray analysis revealed that PKCα was up-regulated by integrin αv in a three-dimensional microenvironment-dependent manner. Integrin αv was observed to promote a relocalization of endogenous p53 from the nucleus to the cytoplasm upon growth in three-dimensional collagen as well as in vivo, whereas stable knockdown of PKCα inhibited the integrin αv-mediated relocalization of p53. Importantly, knockdown of PKCα also promoted apoptosis in three-dimensional collagen and in vivo, resulting in reduced tumor growth. This indicates that PKCα constitutes a crucial component of the integrin αv-mediated pathway(s) that promote p53 relocalization and melanoma survival.     

Collagen XXIII, novel ligand for integrin alpha2beta1 in the epidermis

Cellular receptors for collagens belong to the family of β(1) integrins. In the epidermis, integrin α(2)β(1) is the only collagen-binding integrin present. Its expression is restricted to basal keratinocytes with uniform distribution on the cell surface of those cells. Although α(2)β(1) receptors localized at the basal surface interact with basement membrane proteins collagen IV and laminin 111 and 332, no interaction partners have been reported for these integrin molecules at the lateral and apical membranes of basal keratinocytes. Solid phase binding and surface plasmon resonance spectroscopy demonstrate that collagen XXIII, a member of the transmembrane collagens, directly interacts with integrin α(2)β(1) in an ion- and conformation-dependent manner. The two proteins co-localize on the surface of basal keratinocytes. Furthermore, collagen XXIII is sufficient to induce adhesion and spreading of keratinocytes, a process that is significantly reduced in the absence of functional integrin α(2)β(1).