Natural Compounds As Modulators of Non-apoptotic Cell Death in Cancer Cells.

Cell death is an innate capability of cells to be removed from microenvironment, if and when they are damaged by multiple stresses. Cell death is often regulated by multiple molecular pathways and mechanism, including apoptosis, autophagy, and necroptosis. The molecular network underlying these processes is often intertwined and one pathway can dynamically shift to another one acquiring certain protein components, in particular upon treatment with various drugs. The strategy to treat human cancer ultimately relies on the ability of anticancer therapeutics to induce tumor-specific cell death, while leaving normal adjacent cells undamaged. However, tumor cells often develop the resistance to the drug-induced cell death, thus representing a great challenge for the anticancer approaches. Numerous compounds originated from the natural sources and biopharmaceutical industries are applied today in clinics showing advantageous results. However, some exhibit serious toxic side effects. Thus, novel effective therapeutic approaches in treating cancers are continued to be developed. Natural compounds with anticancer activity have gained a great interest among researchers and clinicians alike since they have shown more favorable safety and efficacy then the synthetic marketed drugs. Numerous studies in vitro and in vivo have found that several natural compounds display promising anticancer potentials. This review underlines certain information regarding the role of natural compounds from plants, microorganisms and sea life forms, which are able to induce non-apoptotic cell death in tumor cells, namely autophagy and necroptosis.

Anticancer Natural Compounds as Epigenetic Modulators of Gene Expression.

Accumulating evidence shows that hallmarks of cancer include: "genetic and epigenetic alterations leading to inactivation of cancer suppressors, overexpression of oncogenes, deregulation of intracellular signaling cascades, alterations of cancer cell metabolism, failure to undergo cancer cell death, induction of epithelial to mesenchymal transition, invasiveness, metastasis, deregulation of immune response and changes in cancer microenvironment, which underpin cancer development". Natural compounds as bioactive ingredients isolated from natural sources (plants, fungi, marine life forms) have revolutionized the field of anticancer therapeutics and rapid developments in preclinical studies are encouraging. Natural compounds could affect the epigenetic molecular mechanisms that modulate gene expression, as well as DNA damage and repair mechanisms. The current review will describe the latest achievements in using naturally produced compounds targeting epigenetic regulators and modulators of gene transcription in vitro and in vivo to generate novel anticancer therapeutics.

Correction: Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation.

[This corrects the article DOI: 10.1371/journal.pone.0136527.].

Dehydroleucodine Induces a TP73-dependent Transcriptional Regulation of Multiple Cell Death Target Genes in Human Glioblastoma Cells.

BACKGROUND: Dehydroleucodine, a natural sesquiterpene lactone from Artemisia douglassiana Besser (Argentine) and Gynoxys verrucosa (Ecuador). OBJECTIVE: To define the molecular mechanisms underlying the effect of dehydroleucodine on the human glioblastoma cells. METHOD: Various techniques (cDNA expression array, real-time quantitative PCR, chromatin immunprecipitation, luciferase reporter assay, use of phosphospecific antibodies, immunoprecipitation, immunoblotting, apoptosis and autophagy assays) were employed to define and validate multiple molecular gene targets affected in human glioblastoma cells upon dehydroleucodine exposure. RESULTS: Dehydroleucodine exposure upregulated the total and phosphorylated (p-Y99) levels of TP73 in U87- MG glioblastoma cells. We found that TP73 silencing led to a partial rescue of U87-MG cells from the cell death induced by dehydroleucodine. Upon the dehydroleucodine exposure numerous gene targets were upregulated and downregulated through a TP73-dependent transcriptional mechanism. Some of these gene targets are known to be involved in cell cycle arrest, apoptosis, autophagy and necroptosis. Dehydroleucodine induced the TP73 binding to the specific genes promoters (CDKN1A, BAX, TP53AIP1, CYLD, RIPK1, and APG5L). Moreover, the exposure of U87-MG cells to dehydroleucodine upregulated the protein levels of CDKN1A, BAX, TP53AIP1, CYLD, RIPK1, APG5L, and downregulated the CASP8 level. The formation of RIPK1 protein complexes and phosphorylation of MLKL were induced by dehydroleucodine supporting the notion of multiple cell death mechanisms implicated in the tumor cell response to dehydroleucodine. CONCLUSION: This multifaceted study led to a conclusion that dehydroleucodine induces the phosphorylation of tumor protein TP73 and in turn activates numerous TP73-target genes regulating apoptosis, autophagy and necroptosis in human glioblastoma cells..

Tumor Protein (TP)-p53 Members as Regulators of Autophagy in Tumor Cells upon Marine Drug Exposure.

Targeting autophagic pathways might play a critical role in designing novel chemotherapeutic approaches in the treatment of human cancers, and the prevention of tumor-derived chemoresistance. Marine compounds were found to decrease tumor cell growth in vitro and in vivo. Some of them were shown to induce autophagic flux in tumor cells. In this study, we observed that the selected marine life-derived compounds (Chromomycin A2, Psammaplin A, and Ilimaquinone) induce expression of several autophagic signaling intermediates in human squamous cell carcinoma, glioblastoma, and colorectal carcinoma cells in vitro through a transcriptional regulation by tumor protein (TP)-p53 family members. These conclusions were supported by specific qPCR expression analysis, luciferase reporter promoter assay, and chromatin immunoprecipitation of promoter sequences bound to the TP53 family proteins, and silencing of the TP53 members in tumor cells.

Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation.

Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites) may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL), a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from Gynoxys verrucosa Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and γ-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment.

Delta Np63 alpha ­ Responsive microRNA Modulate the Expression of Metabolic Enzymes.

MicroRNAs, whose transcription is regulated by members of the tumor protein p53 family, modulate the expression of numerous metabolic enzymes, significantly altering tumor cell response to chemotherapeutic treatments. The role for ΔNp63α-regulated microRNAs in regulation of cell cycle arrest, apoptosis and autophagy in squamous cell carcinoma (SCC) cells upon cisplatin exposure has been reported. The current study indicated that the selected microRNA targets differentially regulated by ΔNp63α in cisplatin-sensitive and cisplatin-resistant SCC cells could alter the expression of a few metabolic enzymes, thereby potentially contributing to the metabolic changes in SCC cells upon cisplatin exposure. Finally, the modulation of specific targets (e.g., SREBF2, AKT2, G6PD, CPS1, FADS1, and ETNK1) using a combination of microRNA mimics and siRNA silencing has shown that a suppression of these metabolic factors/ enzymes could confer a sensitivity of SCC cells to cisplatin. Thus, the Δ Np63α-regulated microRNAs were found to regulate the levels of several metabolic factors and enzymes, thereby potentially contributing to the response of larynx and tongue-derived SCC cells to platinum chemotherapy.

Phospho-ΔNp63α-responsive microRNAs contribute to the regulation of necroptosis in squamous cell carcinoma upon cisplatin exposure.

This study shows that specific microRNAs differentially regulated by ΔNp63α in cisplatin-sensitive and resistant squamous cell carcinoma (SSC) cells of larynx and tongue affect the expression of members of the necroptotic pathway CYLD, RIPK1, and MLKL. Different degrees of protein interaction between necroptotic signaling intermediates were also observed in SCC cells sensitive or resistant to cisplatin. Modulation of RIPK1 with miR-101-3p mimic or inhibitor, as well as with siRNA, or chemical inhibitors was shown to affect sensitivity of SCC cells to cisplatin. This is the first report showing the modulatory effect of ΔNp63α-responsive microRNAs on the specific members of necroptotic pathway in SCC tumor cells variably responding to platinum chemotherapy.

Phospho-ΔNp63α/microRNA network modulates epigenetic regulatory enzymes in squamous cell carcinomas.

The tumor protein (TP) p63/microRNAs functional network may play a key role in supporting the response of squamous cell carcinomas (SCC) to chemotherapy. We show that the cisplatin exposure of SCC-11 cells led to upregulation of miR-297, miR-92b-3p, and miR-485-5p through a phosphorylated ΔNp63α-dependent mechanism that subsequently modulated the expression of the protein targets implicated in DNA methylation (DNMT3A), histone deacetylation (HDAC9), and demethylation (KDM4C). Further studies showed that mimics for miR-297, miR-92b-3p, or miR-485-5p, along with siRNA against and inhibitors of DNMT3A, HDAC9, and KDM4C modulated the expression of DAPK1, SMARCA2, and MDM2 genes assessed by the quantitative PCR, promoter luciferase reporter, and chromatin immunoprecipitation assays. Finally, the above-mentioned treatments affecting epigenetic enzymes also modulated the response of SCC cells to chemotherapeutic drugs, rendering the resistant SCC cells more sensitive to cisplatin exposure, thereby providing the groundwork for novel chemotherapeutic venues in treating patients with SCC.

Phospho-ΔNp63α regulates AQP3, ALOX12B, CASP14 and CLDN1 expression through transcription and microRNA modulation.

Cisplatin-induced and ATM-phosphorylated (p)-ΔNp63α regulates the expression of epidermal differentiation and skin barrier regulators (AQP3, CASP14, ALOX12B, and CLDN1) in squamous cell carcinoma (SCC) cells by dual transcriptional and post-transcriptional mechanisms. We found that p-ΔNp63α bound to target gene promoters, and regulated the activity of the tested promoters in vitro. P-ΔNp63α was shown to upregulate miR-185-5p and downregulate let7-5p, which subsequently modulated AQP3, CASP14, ALOX12B and CLDN1 through their respective 3'-untranslated regions. The introduction of miR-185-5p into resistant SCC-11M cells, which are unable to phosphorylate ΔNp63α, render these cells more sensitive to cisplatin treatment. Further studies of the AQP3, CASP14, ALOX12B, and CLDN1 contributions to chemoresistance may assist in developing novel microRNA-based therapies for human SCC.

Phospho-ΔNp63α-dependent microRNAs modulate chemoresistance of squamous cell carcinoma cells to cisplatin: at the crossroads of cell life and death.

The tumor protein p63/microRNA functional network appears to play a decisive role in chemoresistance of human epithelial cancers. The cisplatin- and phosphorylated-ΔNp63α-dependent microRNAs, whose expression was varied in sensitive and resistant squamous cell carcinoma cells (SCC, which were derived from larynx and tongue tumors), were shown to modulate the expression of multiple members of cell cycle arrest, apoptosis and autophagy pathways. The specific microRNAs were further shown to modulate the resistant phenotype of SCC cells in vitro, thereby providing groundwork for novel chemotherapeutic venues for head and neck cancer.

Phospho-ΔNp63α/microRNA feedback regulation in squamous carcinoma cells upon cisplatin exposure.

Our previous reports showed that the cisplatin exposure induced the ATM-dependent phosphorylation of ΔNp63a, which is subsequently involved in transcriptional regulation of gene promoters encoding mRNAs and microRNAs in squamous cell carcinoma (SCC) cells upon cisplatin-induced cell death. We showed that phosphorylated (p)-ΔNp63a plays a role in upregulation of pro-apoptotic proteins, while non-p-ΔNp63a is implicated in pro-survival signaling. In contrast to non-p-ΔNp63a, p-ΔNp63a modulated expression of specific microRNAs in SCC cells exposed to cisplatin. These microRNAs were shown to attenuate the expression of several proteins involved in cell death/survival, suggesting the critical role for p-ΔNp63a in regulation of tumor cell resistance to cisplatin. Here, we studied the function of ΔNp63a in transcriptional activation and repression of the specific microRNA promoters whose expression is affected by cisplatin treatment of SCC cells. We quantitatively studied chromatin-associated proteins bound to tumor protein (TP) p63-responsive element, we found that p-ΔNp63a along with certain transcription coactivators (e.g., CARM1, KAT2B, TFAP2A, etc.) necessary to induce gene promoters for microRNAs (630 and 885-3p) or with transcription corepressors (e.g., EZH2, CTBP1, HDACs, etc.) needed to repress promoters for microRNAs (181a-5p, 374a-5p and 519a-3p) in SCC cells exposed to cisplatin.

Tumor Protein p63/microRNA Network in Epithelial Cancer Cells.

Non-coding microRNAs are involved in multiple regulatory mechanisms underlying response of cancer cells to stress leading to apoptosis, cell cycle arrest and autophagy. Many molecular layers are implicated in such cellular response including epigenetic regulation of transcription, RNA processing, metabolism, signaling. The molecular interrelationship between tumor protein (TP)-p53 family members and specific microRNAs is a key functional network supporting tumor cell response to chemotherapy and potentially playing a decisive role in chemoresistance of human epithelial cancers. TP63 was shown to modulate the expression of numerous microRNAs involved in regulation of epithelial cell proliferation, differentiation, senescence, "stemness" and skin maintenance, epithelial/ mesenchymal transition, and tumorigenesis in several types of epithelial cancers (e.g. squamous cell carcinoma, ovarian carcinoma, prostate carcinoma, gastric cancer, bladder cancer, and breast tumors), as well as in chemoresistance of cancer cells. TP63/microRNA network was shown to be involved in cell cycle arrest, apoptosis, autophagy, metabolism and epigenetic transcriptional regulation, thereby providing the groundwork for novel chemotherapeutic venues.

Phospho-ΔNp63α/SREBF1 protein interactions: bridging cell metabolism and cisplatin chemoresistance.

Tumor protein (TP)-p53 family members (TP63, TP63 and TP73) are guardians of the genome and key players in orchestrating the cellular response to cisplatin treatment. Cisplatin-induced phosphorylation of ΔNp63α was shown to have a role in regulating intracellular ΔNp63α protein levels. We previously found that squamous cell carcinoma (SCC) cells exposed to cisplatin displayed the ATM-dependent phosphorylation of ΔNp63α (p-ΔNp63α), which is critical for the transcriptional regulation of specific downstream mRNAs and microRNAs and is likely to underlie the chemoresistance of SCC cells. However, SCC cells expressing non-p-ΔNp63α became more cisplatin-resistant. We also found that p-ΔNp63α forms complexes with a number of proteins involved in cell death response through regulation of cell cycle arrest, apoptosis, autophagy, RNA splicing and chromatin modifications. Here, we showed that p-ΔNp63α induced ARG1, GAPDH, and CPT2 gene transcription in cisplatin-sensitive SCC cells, while non-p-ΔNp63α increased a transcription of CAD, G6PD and FASN genes in cisplatin-resistant SCC cells. We report that the p-ΔNp63α-dependent regulatory mechanisms implicated in the modulation of plethora of pathways, including amino acid, carbohydrate, lipid and nucleotide metabolisms, thereby affect tumor cell response to cisplatin-induced cell death, suggesting that the ATM-dependent ΔNp63α pathway plays a role in the resistance of tumor cells to platinum therapy.

A single nucleotide polymorphism in the human PIGK gene associates with low PIGK expression in colorectal cancer patients.

Colorectal cancer (CRC) represents one of the highest incidences of cancers worldwide. Phosphatidylinositol glycan, class K (PIGK), is a crucial member of the glycosyl-phosphatidylinositol transamidase (GPIT) protein complex that attaches a diverse group of macromolecules to the plasma membrane of eukaryotes. However, the precise role of PIGK in tumorigenesis remains largely unknown. Recently, we reported low expression of PIGK protein in primary tumors compared to paired normal tissues of colorectal cancer (CRC) patients. To understand the mechanism underlying this phenomenon, we performed sequencing of all 10 exons of the PIGK gene in 45 CRC patients. Corresponding PIGK protein expression was also evaluated in these patients by immunohistochemistry. No mutation was detected in the coding regions, however, we found a single nucleotide polymorphism (C/C→C/G or G/G; rs1048575) in the 3'UTR of the PIGK gene in 67% (30/45) of the patients. Most of the patients (22/26, 85%) with the altered alleles were of Jewish origin. In comparison, 47% (8/17) of the Arabian patients exhibited the altered C/G alleles. We observed a significantly low (p<0.002) expression of PIGK protein in the patients with the altered alleles (C/G or G/G) compared to the ancestral alleles (C/C). Similarly to the CRC patients, we also examined 5 HCC patients and two HCC cell lines (Hep3B and HepG2) for PIGK genotype (SNP-1048575) and corresponding protein expression. We observed altered alleles (C/G or G/G) and corresponding low PIGK protein expression in 4 out of 5 (80%) primary HCC tumors. Among the HCC cell lines, HepG2 line exhibited ancestral C/C alleles, whereas Hep3B showed altered C/G alleles. Similar to the HCC patients, Hep3B line with the altered alleles (C/G) exhibited significantly low (Student's t-test, p<0.002) PIGK protein expression compared to the Hep3B line carrying the ancestral (C/C) alleles. To examine the exogenous PIGK protein expression status, we transiently transfected both HepG2 (C/C alleles) and Hep3B (C/G alleles) cell lines with wt-PIGK constructs. We detected exogenously expressed PIGK protein in HepG2 (C/C) cells, but no PIGK expression was detectable in Hep3B (C/G) cells at either mRNA or protein level. Our results demonstrate, for the first time, a link between the SNP 1048575 and low PIGK expression in CRC/HCC patients and also suggest a possible association between altered PIGK expression and disease susceptibility.

Global tumor protein p53/p63 interactome: making a case for cisplatin chemoresistance.

Cisplatin chemoresistance is a clinical problem that leads to treatment failure in various human epithelial cancers. Members of tumor protein (TP) p53 family play various critical roles in the multiple molecular mechanisms underlying the chemoresistance of tumor cells. However, the in-depth mechanisms of the cellular response to cisplatin-induced cell death are still under thorough investigation. We previously showed that squamous cell carcinoma (SCC) cells exposed to cisplatin display an ATM-dependent phosphorylation of ΔNp63α, leading to a specific function of the phosphorylated (p)-ΔNp63α transcription factor in cisplatin-sensitive tumor cells. We further found that SCC cells expressing non-p-ΔNp63α-S385G became cisplatin-resistant. Using quantitative mass-spectrometry of protein complexes labeled with isobaric tags, we showed that TP53 and ΔNp63α are involved in numerous protein-protein interactions, which are likely to be implicated in the response of tumor cells to cisplatin exposure. We found that p-ΔNp63α binds to the splicing complex, leading to repression of mRNA splicing and activation of ACIN1-mediated cell death pathway. In contrast to p-ΔNp63α, non-p-ΔNp63α fails to bind the critical members of the splicing complex, thereby leading to activation of RNA splicing and reduction of cell death pathway. Overall, our studies provide an integrated proteomic platform in making a case for the role of the p53/p63 interactome in cisplatin chemoresistance.

Phospho-ΔNp63α-dependent regulation of autophagic signaling through transcription and micro-RNA modulation.

Cisplatin was shown to induce the ataxia telangiectasia mutated (ATM)-dependent phosphorylation of tumor protein p63 isoform, (ΔNp63α), leading to a transcriptional regulation of specific genes implicated in the control of cell death of squamous cell carcinoma (SCC) cells. We previously observed that the cisplatin-induced phosphorylated (p)-ΔNp63α transcriptionally regulates the expression of specific microRNAs (miRNAs) in SCC cells. We found here that cisplatin exposure of SCC cells led to modulation of the members of the autophagic pathway, such as Atg1/Ulk1, Atg3, Atg4A, Atg5, Atg6/Becn1, Atg7, Atg9A and Atg10, by a direct p-ΔNp63α-dependent transcriptional regulation. We further found that specific miRNAs (miR-181a, miR-519a, miR-374a and miR-630), which are critical downstream targets of the p-ΔNp63α, modulated the protein levels of ATG5, ATG6/BECN1, ATG10, ATG12, ATG16L1 and UVRAG, adding another level of expression control for autophagic pathways in SCC cells upon cisplatin exposure. Our data support the notion that the cisplatin-induced p-ΔNp63α could regulate key pathways implicated in response of cancer cells to chemotherapeutics.

Phospho-ΔNp63α/miR-885-3p axis in tumor cell life and cell death upon cisplatin exposure.

The cisplatin-induced ATM-dependent phosphorylated (p)-ΔNp63α plays an important role in transcriptional regulation of specific genes encoding mRNAs and microRNAs (miRs) implicated in cell death, cell survival, and chemoresistance. The p-ΔNp63α-induced miR-885-3p functions as a critical regulator of MDM4, ATK1, BCL2, ATG16L2, ULK2, CASP2, and CASP3 mRNAs via pairing with their respective 'recognition' sequences. Cisplatin exposure modulated the levels of target proteins (reduced BCL2, AKT1, ATG16L2, and ULK2, while activated MDM4) in cisplatin-sensitive wild type ΔNp63α cells leading to distinct changes in cell viability. Finally, miR-885-3p modulated the cisplatin-induced TP53-dependent mitochondrial apoptosis by up regulation of MDM4 levels and down regulation of BCL2 levels in mitochondria. Altogether, our results support the notion that miR-885-3p might contribute in regulation of cell viability, apoptosis and/or autophagy in squamous cell carcinoma cells upon cisplatin exposure.