Pyrazolone-Based Zn(II) Complexes Display Antitumor Effects in Mutant p53-Carrying Cancer Cells.

The synthesis and characterization of nine Schiff bases of pyrazolone ligands HLn (n = 1-9) and the corresponding zinc(II) complexes 1-9 of composition [Zn(Ln)2] (n = 1-9) are reported. The molecular structures of complexes 2, 3, 4, 8, and 9 were determined by single-crystal X-ray diffraction analysis, highlighting in all cases a distorted tetrahedral geometry around the Zn(II) ion. Density functional theory studies are performed on both the HLn ligands and the derived complexes. A mechanism of dissociation and hydrolyzation of the coordinated Schiff base ligands is suggested, confirmed experimentally by powder X-ray diffraction study and photophysical studies. Complexes 1-9 were investigated in vitro as anticancer agents, along with mutant p53 (mutp53) protein levels in human cancer cell lines carrying R175H and R273H mutp53 proteins. Only those complexes with the highest Zn(II) ion release via dissociation have shown a significant cytotoxic activity with reduction of mutp53 protein levels.

HIPK2 in Colon Cancer: A Potential Biomarker for Tumor Progression and Response to Therapies.

Colon cancer, one of the most common and fatal cancers worldwide, is characterized by stepwise accumulation of specific genetic alterations in tumor suppressor genes or oncogenes, leading to tumor growth and metastasis. HIPK2 (homeodomain-interacting protein kinase 2) is a serine/threonine protein kinase and a "bona fide" oncosuppressor protein. Its activation inhibits tumor growth mainly by promoting apoptosis, while its inactivation increases tumorigenicity and resistance to therapies of many different cancer types, including colon cancer. HIPK2 interacts with many molecular pathways by means of its kinase activity or transcriptional co-repressor function modulating cell growth and apoptosis, invasion, angiogenesis, inflammation and hypoxia. HIPK2 has been shown to participate in several molecular pathways involved in colon cancer including p53, Wnt/ß-catenin and the newly identified nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2). HIPK2 also plays a role in tumor-host interaction in the tumor microenvironment (TME) by inducing angiogenesis and cancer-associated fibroblast (CAF) differentiation. The aim of this review is to assess the role of HIPK2 in colon cancer and the underlying molecular pathways for a better understanding of its involvement in colon cancer carcinogenesis and response to therapies, which will likely pave the way for novel colon cancer therapies.

Recent Advances on Mutant p53: Unveiling Novel Oncogenic Roles, Degradation Pathways, and Therapeutic Interventions.

The p53 protein is the master regulator of cellular integrity, primarily due to its tumor-suppressing functions. Approximately half of all human cancers carry mutations in the TP53 gene, which not only abrogate the tumor-suppressive functions but also confer p53 mutant proteins with oncogenic potential. The latter is achieved through so-called gain-of-function (GOF) mutations that promote cancer progression, metastasis, and therapy resistance by deregulating transcriptional networks, signaling pathways, metabolism, immune surveillance, and cellular compositions of the microenvironment. Despite recent progress in understanding the complexity of mutp53 in neoplastic development, the exact mechanisms of how mutp53 contributes to cancer development and how they escape proteasomal and lysosomal degradation remain only partially understood. In this review, we address recent findings in the field of oncogenic functions of mutp53 specifically regarding, but not limited to, its implications in metabolic pathways, the secretome of cancer cells, the cancer microenvironment, and the regulating scenarios of the aberrant proteasomal degradation. By analyzing proteasomal and lysosomal protein degradation, as well as its connection with autophagy, we propose new therapeutical approaches that aim to destabilize mutp53 proteins and deactivate its oncogenic functions, thereby providing a fundamental basis for further investigation and rational treatment approaches for TP53-mutated cancers.

NRF2 activation in BON­1 neuroendocrine cancer cells reduces the cytotoxic effects of a novel Ruthenium(II)­curcumin compound: A pilot study.

Gastroenteropancreatic neuroendocrine neoplasms (GEP­NEN) are a group of rare tumors whose specific pathogenetic mechanisms of resistance to therapies have not been completely revealed yet. Chemotherapy is the main therapeutic approach in patients with GEP­NEN, however, novel combination regimens and targeted therapy are continuously explored. In the present study, the anticancer effect of a novel Ruthenium (Ru)(II)­Bisdemethoxycurcumin (Ru­bdcurc) compound was evaluated in BON­1 cell line, one of the few cell lines derived from GEP­NEN, largely used in experimental research of this type of tumors. The experimental data revealed that the Ru­bdcurc compound induced cell death in a dose­dependent manner, in vitro. Biochemical studies demonstrated that, in response to the lower dose of treatment, BON­1 cells activated the nuclear factor erythroid 2­related factor 2 (NRF2) pathway with induction of some of its targets including catalase and p62 as well as of the antiapoptotic marker Bcl2, all acting as chemoresistance mechanisms. NRF2 induction associated also with increased expression of endogenous p53 which is reported to be dysfunctional in BON­1 cells and to inhibit apoptosis. Genetic or pharmacologic targeting of NRF2 inhibited the activation of the NRF2 pathway, as well as of endogenous dysfunctional p53, in response to the lower dose of Ru­bdcurc, increasing the cell death. To assess the interplay between NRF2 and dysfunctional p53, genetic targeting of p53 showed reduced activation of the NRF2 pathway in response to the lower dose of Ru­bdcurc, increasing the cell death. These findings identified for the first time a possible dysfunctional p53/NRF2 interplay in BON­1 cell line that can be a novel key determinant in cell resistance to cytotoxic agents to be evaluated also in GEP­NEN.

New Copper-Based Metallodrugs with Anti-Invasive Capacity.

While metal-based complexes are deeply investigated as anticancer chemotherapeutic drugs, fewer studies are devoted to their anti-invasive activity. Herein, two copper (Cu)(II) tropolone derivatives, [Cu(Trop)Cl] and [Cu(Trop)Sac], both containing the N,N-chelated 4,4'-bishydroxymethyl-2,2'-bipyridne ligand, were evaluated for their anticancer and anti-invasive properties. RKO (RKO-ctr) colon cancer cells and their derivatives undergoing stable small interference (si) RNA for HIPK2 protein (RKO-siHIPK2) with acquisition of pro-invasive capacity were used. The results demonstrate that while [Cu(Trop)Sac] did not show cytotoxic activity, [Cu(Trop)Cl] induced cell death in both RKO-ctr and RKO-siHIPK2 cells, indicating that structural changes on substituting the coordinated chloride ligand with saccharine (Sac) could be a key factor in suppressing mechanisms of cellular death. On the other hand, both [Cu(Trop)Sac] and [Cu(Trop)Cl] complexes counteracted RKO-siHIPK2 cell migration in the wound healing assay. The synergic effect exerted by the concomitant presence of both tropolone and saccharin ligands in [Cu(Trop)Sac] was also supported by its significant inhibition of RKO-siHIPK2 cell migration compared to the free Sac ligand. These data suggest that the two Cu(II) tropolone derivatives are also interesting candidates to be further tested in in vivo models as an anti-invasive tumor strategy.

The Sweet Side of HIPK2.

HIPK2 is an evolutionary conserved protein kinase which modulates many molecular pathways involved in cellular functions such as apoptosis, DNA damage response, protein stability, and protein transcription. HIPK2 plays a key role in the cancer cell response to cytotoxic drugs as its deregulation impairs drug-induced cancer cell death. HIPK2 has also been involved in regulating fibrosis, angiogenesis, and neurological diseases. Recently, hyperglycemia was found to positively and/or negatively regulate HIPK2 activity, affecting not only cancer cell response to chemotherapy but also the progression of some diabetes complications. The present review will discuss how HIPK2 may be influenced by the high glucose (HG) metabolic condition and the consequences of such regulation in medical conditions.

HIPK2 in Angiogenesis: A Promising Biomarker in Cancer Progression and in Angiogenic Diseases.

Angiogenesis is the formation of new blood capillaries taking place from preexisting functional vessels, a process that allows cells to cope with shortage of nutrients and low oxygen availability. Angiogenesis may be activated in several pathological diseases, from tumor growth and metastases formation to ischemic and inflammatory diseases. New insights into the mechanisms that regulate angiogenesis have been discovered in the last years, leading to the discovery of new therapeutic opportunities. However, in the case of cancer, their success may be limited by the occurrence of drug resistance, meaning that the road to optimize such treatments is still long. Homeodomain-interacting protein kinase 2 (HIPK2), a multifaceted protein that regulates different molecular pathways, is involved in the negative regulation of cancer growth, and may be considered a "bona fide" oncosuppressor molecule. In this review, we will discuss the emerging link between HIPK2 and angiogenesis and how the control of angiogenesis by HIPK2 impinges in the pathogenesis of several diseases, including cancer.

NRF2 and Bip Interconnection Mediates Resistance to the Organometallic Ruthenium-Cymene Bisdemethoxycurcumin Complex Cytotoxicity in Colon Cancer Cells.

Organometallic ruthenium (Ru)(II)-cymene complexes display promising pharmacological properties and might represent alternative therapeutic agents in medical applications. Polyphenols, such as curcumin and curcuminoids, display beneficial properties in medicine, including chemoprevention. Here we analyzed the anticancer effect of a cationic Ruthenium (Ru)(II)-cymene Bisdemethoxycurcumin (Ru-bdcurc) complex. The experimental data show that Ru-bdcurc induced cell death of colon cancer cells in vitro. In response to treatment, cancer cells activated the endoplasmic reticulum (ER)-resident chaperone GRP78/BiP and NRF2, the master regulators of the unfolded protein response (UPR) and the antioxidant response, respectively. Pharmacologic targeting of either NRF2 or BiP potentiated the cytotoxic effect of Ru-bdcurc. We also found that NRF2 and UPR pathways were interconnected as the inhibition of NRF2 reduced BiP protein levels. Mechanistically, the increased Ru-bdcurc-induced cell death, following NRF2 or BiP inhibition, correlated with the upregulation of the UPR apoptotic marker CHOP and with increased H2AX phosphorylation, a marker of DNA damage. The findings reveal that BiP and NRF2 interconnection was a key regulator of colon cancer cells resistance to Ru-bdcurc cytotoxic effect. Targeting that interconnection overcame the protective mechanism and enhanced the antitumor effect of the Ru-bdcurc compound.

HIPK2 as a Novel Regulator of Fibrosis.

Fibrosis is an unmet medical problem due to a lack of evident biomarkers to help develop efficient targeted therapies. Fibrosis can affect almost every organ and eventually induce organ failure. Homeodomain-interacting protein kinase 2 (HIPK2) is a protein kinase that controls several molecular pathways involved in cell death and development and it has been extensively studied, mainly in the cancer biology field. Recently, a role for HIPK2 has been highlighted in tissue fibrosis. Thus, HIPK2 regulates several pro-fibrotic pathways such as Wnt/ß-catenin, TGF-ß and Notch involved in renal, pulmonary, liver and cardiac fibrosis. These findings suggest a wider role for HIPK2 in tissue physiopathology and highlight HIPK2 as a promising target for therapeutic purposes in fibrosis. Here, we will summarize the recent studies showing the involvement of HIPK2 as a novel regulator of fibrosis.

The Impact of NRF2 Inhibition on Drug-Induced Colon Cancer Cell Death and p53 Activity: A Pilot Study.

Nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2) protein is the master regulator of oxidative stress, which is at the basis of various chronic diseases including cancer. Hyperactivation of NRF2 in already established cancers can promote cell proliferation and resistance to therapies, such as in colorectal cancer (CRC), one of the most lethal and prevalent malignancies in industrialized countries with limited patient overall survival due to its escape mechanisms in both chemo- and targeted therapies. In this study, we generated stable NRF2 knockout colon cancer cells (NRF2-Cas9) to investigate the cell response to chemotherapeutic drugs with regard to p53 oncosuppressor, whose inhibition we previously showed to correlate with NRF2 pathway activation. Here, we found that NRF2 activation by sulforaphane (SFN) reduced cisplatin (CDDP)-induced cell death only in NRF2-proficient cells (NRF2-ctr) compared to NRF2-Cas9 cells. Mechanistically, we found that NRF2 activation protected NRF2-ctr cells from the drug-induced DNA damage and the apoptotic function of the unfolded protein response (UPR), in correlation with reduction of p53 activity, effects that were not observed in NRF2-Cas9 cells. Finally, we found that ZnCl2 supplementation rescued the cisplatin cytotoxic effects, as it impaired NRF2 activation, restoring p53 activity. These findings highlight NRF2's key role in neutralizing the cytotoxic effects of chemotherapeutic drugs in correlation with reduced DNA damage and p53 activity. They also suggest that NRF2 inhibition could be a useful strategy for efficient anticancer chemotherapy and support the use of ZnCl2 to inhibit NRF2 pathway in combination therapies.

P62/SQSTM1/Keap1/NRF2 Axis Reduces Cancer Cells Death-Sensitivity in Response to Zn(II)-Curcumin Complex.

The hyperactivation of nuclear factor erythroid 2 p45-related factor 2 (NRF2), frequently found in many tumor types, can be responsible for cancer resistance to therapies and poor patient prognosis. Curcumin has been shown to activate NRF2 that has cytotprotective or protumorigenic roles according to tumor stage. The present study aimed at investigating whether the zinc-curcumin Zn(II)-curc compound, which we previously showed to display anticancer effects through multiple mechanisms, could induce NRF2 activation and to explore the underlying molecular mechanisms. Biochemical studies showed that Zn(II)-curc treatment increased the NRF2 protein levels along with its targets, heme oxygenase-1 (HO-1) and p62/SQSTM1, while markedly reduced the levels of Keap1 (Kelch-like ECH-associated protein 1), the NRF2 inhibitor, in the cancer cell lines analyzed. The silencing of either NRF2 or p62/SQSTM1 with specific siRNA demonstrated the crosstalk between the two molecules and that the knockdown of either molecule increased the cancer cell sensitivity to Zn(II)-curc-induced cell death. This suggests that the crosstalk between p62/SQSTM1 and NRF2 could be therapeutically exploited to increase cancer patient response to therapies.

A ruthenium(II)-curcumin compound modulates NRF2 expression balancing the cancer cell death/survival outcome according to p53 status.

BACKGROUND: Tumor progression and tumor response to anticancer therapies may be affected by activation of oncogenic pathways such as the antioxidant one induced by NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor and the pathways modified by deregulation of oncosuppressor p53. Often, oncogenic pathways may crosstalk between them increasing tumor progression and resistance to anticancer therapies. Therefore, understanding that interplay is critical to improve cancer cell response to therapies. In this study we aimed at evaluating NRF2 and p53 in several cancer cell lines carrying different endogenous p53 status, using a novel curcumin compound since curcumin has been shown to target both NRF2 and p53 and have anti-tumor activity. METHODS: We performed biochemical and molecular studies by using pharmacologic of genetic inhibition of NRF2 to evaluate the effect of curcumin compound in cancer cell lines of different tumor types bearing wild-type (wt) p53, mutant (mut) p53 or p53 null status. RESULTS: We found that the curcumin compound induced a certain degree of cell death in all tested cancer cell lines, independently of the p53 status. At molecular level, the curcumin compound induced NRF2 activation, mutp53 degradation and/or wtp53 activation. Pharmacologic or genetic NRF2 inhibition further increased the curcumin-induced cell death in both mutp53- and wtp53-carrying cancer cell lines while it did not increase cell death in p53 null cells, suggesting a cytoprotective role for NRF2 and a critical role for functional p53 to achieve an efficient cancer cell response to therapy. CONCLUSIONS: These findings underline the prosurvival role of curcumin-induced NRF2 expression in cancer cells even when cells underwent mutp53 downregulation and/or wtp53 activation. Thus, NRF2 inhibition increased cell demise particularly in cancer cells carrying p53 either wild-type or mutant suggesting that p53 is crucial for efficient cancer cell death. These results may represent a paradigm for better understanding the cancer cell response to therapies in order to design more efficient combined anticancer therapies targeting both NRF2 and p53.

Nuclear factor erythroid 2 (NF-E2) p45-related factor 2 interferes with homeodomain-interacting protein kinase 2/p53 activity to impair solid tumors chemosensitivity.

Resistance to chemotherapy represents a major hurdle to successful cancer treatment. A key role for efficient response to anticancer therapies is played by TP53 oncosuppressor gene that indeed is mutated in 50% of human cancers or inactivated at protein level in the remaining 50%. Homeodomain-interacting protein kinase 2 (HIPK2) is the wild-type p53 (wtp53) apoptotic activator, and its inhibition by hypoxia or hyperglycemia may contribute to tumor chemoresistance mainly by impairing p53 apoptotic activity. Another important molecule able to induce chemoresistance is nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2) transcription factor, whose activation by oxidative and/or electrophilic stress regulates a transcriptional antioxidant program allowing cancer cells to adapt and survive to stresses. NRF2 may shift from cytoprotective to tumor-promoting function, according to tumor phases. NRF2 may crosstalk with both wtp53 and mutant p53 (mutp53), inhibiting the wtp53 apoptotic function and strengthening the mutp53 oncogenic function. NRF2 has also been shown to induce HIPK2 mRNA expression cooperating in inducing cytoprotection. Although HIPK2, p53, and NRF2 have been individually extensively studied, their interplay has not been clearly addressed yet. On the basis of the background and our results, we aim at hypothesizing the unexpected pro-survival activity played by the NRF2/HIPK2/p53 interplay that can be hijacked by cancer cells to bypass drugs cytotoxicity.

PBA Preferentially Impairs Cell Survival of Glioblastomas Carrying mutp53 by Reducing Its Expression Level, Stabilizing wtp53, Downregulating the Mevalonate Kinase and Dysregulating UPR.

Phenylbutyrate (PBA) is a derivative of Butyric Acid (BA), which has the characteristics of being a histone deacetylase (HDAC) inhibitor and acting as a chemical chaperone. It has the potential to counteract a variety of different diseases, from neurodegeneration to cancer. In this study, we investigated the cytotoxic effect of PBA against glioblastoma cells carrying wt or mutant (mut) p53 and found that it exerted a higher cytotoxic effect against the latter in comparison with the former. This could be due to the downregulation of mutp53, to whose pro-survival effects cancer cells become addicted. In correlation with mutp53 reduction and wtp53 activation, PBA downregulated the expression level of mevalonate kinase (MVK), a key kinase of the mevalonate pathway strongly involved in cancer cell survival. Here we differentiated the chaperoning function of PBA from the others anti-cancer potentiality by comparing its effects to those exerted by NaB, another HDACi that derives from BA but, lacking the phenyl group, cannot act as a chemical chaperone. Interestingly, we observed that PBA induced a stronger cytotoxic effect compared to NaB against U373 cells as it skewed the Unfolded Protein Response (UPR) towards cell death induction, upregulating CHOP and downregulating BIP, and was more efficient in downregulating MVK. The findings of this study suggest that PBA represents a promising molecule against glioblastomas, especially those carrying mutp53, and its use, approved by FDA for urea cycle disorders, should be extended to the glioblastoma anticancer therapy.

Interplay between Endoplasmic Reticulum (ER) Stress and Autophagy Induces Mutant p53H273 Degradation.

The unfolded protein response (UPR) is an adaptive response to intrinsic and external stressors, and it is mainly activated by the accumulation of misfolded proteins at the endoplasmic reticulum (ER) lumen producing ER stress. The UPR signaling network is interconnected with autophagy, the proteolytic machinery specifically devoted to clearing misfolded proteins in order to survive bioenergetic stress and/or induce cell death. Oncosuppressor TP53 may undergo inactivation following missense mutations within the DNA-binding domain (DBD), and mutant p53 (mutp53) proteins may acquire a misfolded conformation, often due to the loss of the DBD-bound zinc ion, leading to accumulation of hyperstable mutp53 proteins that correlates with more aggressive tumors, resistance to therapies, and poorer outcomes. We previously showed that zinc supplementation induces mutp53 protein degradation by autophagy. Here, we show that mutp53 (i.e., Arg273) degradation following zinc supplementation is correlated with activation of ER stress and of the IRE1α/XBPI arm of the UPR. ER stress inhibition with chemical chaperone 4-phenyl butyrate (PBA) impaired mutp53 downregulation, which is similar to IRE1α/XBPI specific inhibition, reducing cancer cell death. Knockdown of mutp53 failed to induce UPR/autophagy activation indicating that the effect of zinc on mutp53 folding was likely the key event occurring in ER stress activation. Recently discovered small molecules targeting components of the UPR show promise as a novel anticancer therapeutic intervention. However, our findings showing UPR activation during mutp53 degradation indicate that caution is necessary in the design of therapies that inhibit UPR components.

Reduced chemotherapeutic sensitivity in high glucose condition: implication of antioxidant response.

Resistance to chemotherapy represents a major obstacle to successful treatment. The generation of reactive oxygen species (ROS) has been directly linked to the cytotoxic effects of several antitumor agents, including Adriamycin (ADR), and modulation of the oxidative balance has been implicated in the development and/or regulation of resistance to chemotherapeutic drugs. We recently showed that high glucose (HG) markedly diminished the cancer cell death induced by anticancer agents such as ADR. In the present study we attempted to evaluate the mechanism that impaired the cytotoxic effect of ADR in HG. We found that, in colon cancer cells, HG attenuated ADR-induced ROS production that consequently diminished ADR-induced H2AX phosphorylation and micronuclei (MN) formation. Mechanistically, HG attenuation of ADR-induced ROS production correlated with increased antioxidant response promoted by NRF2 activity. Thus, pharmacologic inhibition of NRF2 pathway by brusatol re-established the ADR cytotoxic effect impaired by HG. Together, the data provide new insights into chemotherapeutic-resistance mechanisms in HG condition dictated by increased NRF2-induced antioxidant response and how they may be overcome in order to restore chemosensitivity and ADR-induced cell death.

HIPK2 role in the tumor-host interaction: Impact on fibroblasts transdifferentiation CAF-like.

The dialogue between cancer cells and the surrounding fibroblasts, tumor-associated macrophages (TAM), and immune cells can create a tumor microenvironment (TME) able to promote tumor progression and metastasis and induce resistance to anticancer therapies. Cancer cells, by producing growth factors and cytokines, can recruit and activate fibroblasts in the TME inducing their transdifferention in cancer-associated fibroblasts (CAFs). Then, CAFs, in a reciprocal cross-talk with cancer cells, sustain cancer growth and survival and support malignancy and tumor resistance to therapies. Therefore, the identification of the molecular mechanisms regulating the interplay between cancer cells and fibroblasts can offer an intriguing opportunity for novel diagnostic and therapeutic anticancer purpose. HIPK2 is a multifunctional tumor suppressor protein that modulates cancer cell growth and apoptosis in response to anticancer drugs and negatively regulates pathways involved in tumor progression and chemoresistance. HIPK2 protein downregulation is induced by hypoxia and hyperglycemia and HIPK2 knockdown favors tumor progression and resistance to therapy other than a pseudohypoxic, inflammatory, and angiogenic cancer phenotype. Therefore, we hypothesized that HIPK2 modulation in cancer cells could contribute to modify the tumor-host interaction. In support of our hypothesis, here we provide evidence that culturing human fibroblasts (hFB) with conditioned media derived from cancer cells undergoing HIPK2 knockdown (CMsiHIPK2 ) triggered their transdifferentiation CAF-like, compared to hFB cultured with CM-derived from HIPK2-carrying control cancer cells. CAF transdifferentiation was identified by expression of several markers including α-smooth muscle actin (α-SMA) and collagen I and correlated with autophagy-mediated caveolin-1 degradation. Although the molecular mechanisms dictating CAF-transdifferentiation need to be elucidated, these results open the way to further study the role of HIPK2 in TME remodeling for prognostic and therapeutic purpose.

Autophagy manipulation as a strategy for efficient anticancer therapies: possible consequences.

Autophagy is a catabolic process whose activation may help cancer cells to adapt to cellular stress although, in some instances, it can induce cell death. Autophagy stimulation or inhibition has been considered an opportunity to treat cancer, especially in combination with anticancer therapies, although autophagy manipulation may be viewed as controversial. Thus, whether to induce or to inhibit autophagy may be the best option in the different cancer patients is still matter of debate. Her we will recapitulate the possible advantages or disadvantages of manipulating autophagy in cancer, not only with the aim to obtain cancer cell death and disable oncogenes, but also to evaluate its interplay with the immune response which is fundamental for the success of anticancer therapies.

p53-Dependent PUMA to DRAM antagonistic interplay as a key molecular switch in cell-fate decision in normal/high glucose conditions.

BACKGROUND: As an important cellular stress sensor phosphoprotein p53 can trigger cell cycle arrest and apoptosis and regulate autophagy. The p53 activity mainly depends on its transactivating function, however, how p53 can select one or another biological outcome is still a matter of profound studies. Our previous findings indicate that switching cancer cells in high glucose (HG) impairs p53 apoptotic function and the transcription of target gene PUMA. METHODS AND RESULTS: Here we report that, in response to drug adriamycin (ADR) in HG, p53 efficiently induced the expression of DRAM (damage-regulated autophagy modulator), a p53 target gene and a stress-induced regulator of autophagy. We found that ADR treatment of cancer cells in HG increased autophagy, as displayed by greater LC3II accumulation and p62 degradation compared to ADR-treated cells in low glucose. The increased autophagy in HG was in part dependent on p53-induced DRAM; indeed DRAM knockdown with specific siRNA reversed the expression of the autophagic markers in HG. A similar outcome was achieved by inhibiting p53 transcriptional activity with pifithrin-α. DRAM knockdown restored the ADR-induced cell death in HG to the levels obtained in low glucose. A similar outcome was achieved by inhibition of autophagy with cloroquine (CQ) or with silencing of autophagy gene ATG5. DRAM knockdown or inhibition of autophagy were both able to re-induce PUMA transcription in response to ADR, underlining a reciprocal interplay between PUMA to DRAM to unbalance p53 apoptotic activity in HG. Xenograft tumors transplanted in normoglycemic mice displayed growth delay after ADR treatment compared to those transplanted in diabetics mice and such different in vivo response correlated with PUMA to DRAM gene expression. CONCLUSIONS: Altogether, these findings suggest that in normal/high glucose condition a mutual unbalance between p53-dependent apoptosis (PUMA) and autophagy (DRAM) gene occurred, modifying the ADR-induced cancer cell death in HG both in vitro and in vivo.

Oxidant species are involved in T/B-mediated ERK1/2 phosphorylation that activates p53-p21 axis to promote KSHV lytic cycle in PEL cells.

KSHV is a gammaherpesvirus strongly associated to human cancers such as Primary Effusion Lymphoma (PEL) and Kaposi's Sarcoma. The naturally virus-infected tumor cells usually display latent infection since a minority of cells undergoes spontaneous viral replication. The lytic cycle can be induced in vitro upon appropriate stimuli such as TPA (T), alone or in combination with butyrate (B), (T/B). In previous studies, Protein Kinase C (PKC) δ, Extracellular Signal-regulated Kinase1/2 (ERK1/2) and p53-p21 axis have been separately reported to play a role in KSHV reactivation from latency. Here, we found that these pathways were interconnected to induce KSHV lytic cycle in PEL cells treated with T/B. T/B also increased H2O2 that played an important role in the activation of these pathways. Oxidant specie production correlated with PKC δ activation, as the PKC δ inhibitor rottlerin reduced both H2O2 and KSHV lytic antigen expression. H2O2 contributed to T/B-mediated ERK1/2 activation that mediated p53 phosphorylation at serine 15 (Ser15) and increased p21 expression. Oxidant specie inhibition by quercetin indeed strongly reduced the activation of these pathways, lytic antigen expression and interestingly it also increased T/B-induced cell death. The use of ERK inhibitor PD98059 or p53 silencing demonstrated the importance of p53Ser15 phosphorylation and of p53-p21 axis in KSHV lytic cycle activation. Understanding the role of oxidant species and the molecular mechanisms involved in KSHV lytic cycle induction is particularly important since oxidant species represent the most physiological stimulus for viral reactivation in vivo and it is known that viral production contributes to the maintenance/progression of KSHV associated malignancies.