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From the perspective of circular economy, it is extremely useful to recycle waste products for human health applications. Among the health-beneficial properties of bioactive phyto-compounds, grape pomace represents a precious source of bioactive molecules with potential antitumor properties. Here, we describe the effects of a Sicilian grape pomace hydroalcoholic extract (HE) in colon and breast cancer cells. The characterization of HE composition revealed the predominance of anthoxanthins and phenolic acids. HE treatment was more effective in reducing the viability of colon cancer cells, while breast cancer cells appeared more resistant. Indeed, while colon cancer cells underwent apoptosis, as shown by DNA fragmentation, caspase-3 activation, and PARP1 degradation, breast cancer cells seemed to not undergo apoptosis. To elucidate the underlying mechanisms, reactive oxygen species (ROS) were evaluated. Interestingly, ROS increased in both cell lines but, while in colon cancer, cells' ROS rapidly increased and progressively diminished over time, in breast cancer, cells' ROS increase was persistent up to 24 h. This effect was correlated with the induction of pro-survival autophagy, demonstrated by autophagosomes formation, autophagic markers increase, and protection by the antioxidant NAC. The autophagy inhibitor bafilomycin A1 significantly increased the HE effects in breast cancer cells but not in colon cancer cells. Overall, our data provide evidence that HE efficacy in tumor cells depends on a balance between ROS-mediated autophagy and apoptosis. Therefore, inhibiting pro-survival autophagy may be a tool to target those cells that appear more resistant to the effect of HE.

Litchi chinensis Sonnerat is a tropical tree whose fruits contain significant amounts of bioactive polyphenols. Litchi cultivation has recently spread in Sicily where the climate conditions are particularly favorable for this crop. Recent findings have shown that Litchi extracts display anti-tumor and pro-apoptotic effects in vitro, but the precise underlying mechanisms have not been fully elucidated. In this study, we report for the first time the effects of Sicilian litchi fruit extracts on colon cancer cells. The results indicated that litchi exocarp, mesocarp and endocarp fractions reduce the viability and clonogenic growth of HT29 cells. These effects were due to cell cycle arrest in the G2/M phase followed by caspase-dependent cell death. Interestingly, litchi exocarp and endocarp triggered a precocious autophagic response (16⁻24 h), which was accompanied by an increase in the level of autophagy related 1/autophagy activating kinase 1 (ATG1/ULK1), beclin-1, microtubule associated protein 1 light chain 3 (LC3)-II and p62 proteins. Autophagy inhibition by bafilomycin A1 or beclin-1 silencing increased cell death, thus suggesting that autophagy was initially triggered as a pro-survival response. Significant effects of Litchi extracts were also observed in other colon cancer cells, including HCT116 and Caco-2 cells. On the other hand, differentiated Caco-2 cells, a model of human enterocytes, appeared to be insensitive to the extracts at the same treatment conditions. High-Performance Liquid Chromatography⁻Electrospray Ionization-Quadrupole-Time-Of-Flight HPLC/ESI/Q-TOF evidenced the presence of some polyphenolic compounds, specifically in exocarp and endocarp extracts, that can account for the observed biological effects. The results obtained suggest a potential therapeutic efficacy of polyphenolic compounds purified from Sicilian Litchi fractions for the treatment of colon cancer. Moreover, our findings indicate that modulation of autophagy can represent a tool to improve the effectiveness of these agents and potentiate the anti-tumor response of colon cancer cells.

The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However, when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in Δψm, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy., (© 2014 Wiley Periodicals, Inc.)

Retinoblastoma is the most common intraocular malignancy of childhood. In developing countries, treatment is limited, long-term survival rates are low and current chemotherapy causes significant morbidity to pediatric patients and significantly limits dosing. Therefore there is an urgent need to identify new therapeutic strategies to improve the clinical outcome of patients with retinoblastoma. Here, we investigated the effects of two natural compounds okadaic acid (OKA) and parthenolide (PN) on human retinoblastoma Y79 cells. For the first time we showed that OKA/PN combination at subtoxic doses induces potent synergistic apoptotic effects accompanied by lowering in p-Akt levels, increasing in the stabilized forms of p53 and potent decrease in pS166-Mdm2. We also showed the key involvement of PTEN which, after OKA/PN treatment, potently increased before p53, thus suggesting that p53 activation was under PTEN action. Moreover, after PTEN-knockdown p-Akt/ pS166Mdm2 increased over basal levels and p53 significantly lowered, while OKA/PN treatment failed both to lower p-Akt and pS166-Mdm2 and to increase p53 below/over their basal levels respectively. OKA/PN treatment potently increased ROS levels whereas decreased those of GSH. Reducing cellular GSH by l-butathionine-[S,R]-sulfoximine treatment significantly anticipated the cytotoxic effect exerted by OKA/PN. Furthermore, the effects of OKA/PN treatment on both GSH content and cell viability were less pronounced in PTEN silenced cells than in control cells. The results provide strong suggestion for combining a treatment approach that targets the PTEN/Akt/Mdm2/p53 pathway.

Paclitaxel (PTX) and beta-lapachone (LPC) are naturally occurring compounds that have shown a large spectrum of anticancer activity. In this article we show for the first time that PTX/LPC combination induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and LPC (1.5 microM) caused biochemical and morphological signs of apoptosis at 48 h of treatment. These effects were accompanied by potent lowering in inhibitor of apoptosis proteins and by activation of Bid and caspases 3 and 6 with lamin B and PARP breakdown. PTX/LPC combination acted by favoring p53 stabilization through a lowering in p-Akt levels and in ps166-MDM2, the phosphorylated-MDM2 form that enters the nucleus and induces p53 export and degradation. Treatment with wortmannin or transfection with a dominant negative form of Akt anticipated at 24 h the effects induced by PTX/LPC, suggesting a protective role against apoptosis played by Akt in Y79 cells. In line with these results, we demonstrated that Y79 cells contain constitutively active Akt, which forms a cytosolic complex with p53 and MDM2 driving p53 degradation. PTX/LPC treatment induced a weakness of Akt-MDM2-p53 complex and increased nuclear p53 levels. Our results suggest that phospho-Akt lowering is at the root of the apoptotic action exerted by PTX/LPC combination and provide strong validation for a treatment approach that targets survival signals represented by phospho-Akt and inhibitor of apoptosis proteins., ((c) 2009 Wiley-Liss, Inc.)

This paper shows that the histone deacetylase inhibitor SAHA sensitised at sub-toxic doses human hepatocellular carcinoma cells (HepG2, Hep3B and SK-Hep1) to TRAIL-induced apoptosis, while it was ineffective in primary human hepatocytes (PHHs). In particular in HCC cells SAHA increased the expression of death receptor 5 (DR5) and caused a decrement of c-Flip. These two modifications provoked in the presence of TRAIL the rapid production of TRAIL-DISC and the activation of caspase-8. Consequently SAHA/TRAIL combination induced many apoptotic events, such as a cleavage of Bid into tBid, dissipation of mitochondrial membrane potential, activation of caspase-3 with the consequent cleavage of both NF-kB and Akt. The decrease in NF-kB level seemed to be responsible for the reduction in the content of IAP family antiapoptotic proteins while the decrease in Akt level caused a reduction in phospho-Bad. These events led to the activation of caspase-9, which contributed to the strong apoptotic activity of TRAIL. Sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by SAHA may suggest new strategies for the treatment of liver tumours.

This report shows that histone deacetylase inhibitors (HDACIs) induced apoptosis in human hepatoma HepG2 cells in a dose- and time-dependent manner. Trichostatin A (TSA), ITF2357 and suberoylanilide hydroxamic acid (SAHA), which were very effective agents, caused apoptotic effects after a lag phase of 12-16 h. In order to elucidate the mechanism of HDACIs action in HepG2 cells we have studied the effects of TSA, ITF2357 and SAHA on acetylation of p53 and histones H2A, H2B, H3 and H4. It was observed that HDACIs rapidly induced acetylation of these proteins, being the effects clearly visible already at 30 min of treatment at the same doses which caused apoptosis. Analysis of the immunocomplexes, obtained from nuclear extracts using an antibody against p53, revealed the presence of acetylated p53 together with acetylated forms of histones and histone acetyltransferases p300 and PCAF. Experiments performed using pifithrin-alpha, a reversible inhibitor of p53, showed a correlation between acetylation of p53 and induction of apoptosis. In addition treatment with siRNA against p53 indicated that p53 is involved in the acetylation of histones. In conclusion, this report suggests that complexes constituted by acetylated p53, acetylated histones and coactivators can play a central role in HDACI-induced apoptosis in HepG2 cells.

Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. This paper shows that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) stimulated at 5-10 microM apoptosis in human hepatoma HepG2 and Huh6 cells, but was ineffective in primary human hepatocytes (PHH). In HepG2 cells SAHA induced the extrinsic apoptotic pathway, increasing the expression of both FasL and FasL receptor and causing the activation of caspase-8. Moreover, SAHA enhanced the level of Bim proteins, stimulated alternative splicing of the Bcl-X transcript with the expression of the proapoptotic Bcl-Xs isoform, induced degradation of Bid into the apoptotic factor t-Bid and dephosphorylation and inactivation of the anti-apoptotic factor Akt. Consequently, SAHA caused loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, activation of caspase-3 and degradation of PARP. Interestingly, a combination of suboptimal doses of SAHA (1 microM) and bortezomib (5-10 nM), a potent inhibitor of 26S proteasome, synergistically induced apoptosis in both HepG2 and Huh6 cells, but was ineffective in PHH. Combined treatment increased with synergistic effects the expression levels of c-Jun, phospho-c-Jun and FasL and the production of Bcl-Xs. These effects were accompanied by activation of Bid, caspase-8 and 3. In conclusion, SAHA stimulated apoptosis in hepatoma cells and exerted a synergistic apoptotic effect when combined with bortezomib. In contrast, these treatments were quite ineffective in inducing apoptosis in PHH. Thus, our results suggest the potential application of the SAHA/bortezomib combination in clinical trials for liver cancer.

The proteasome inhibitor bortezomib is an efficacious apoptotic agent in many tumor cells. This paper shows that bortezomib induced apoptosis in human hepatoma HepG2 cells associated with many modifications in the expression of survival or death factors. Although bortezomib increased the level of the protective factors HSP70 and HSP27, the effects of the drug that favour cell death were predominant. These events include accumulation of c-Jun, phospho-c-Jun and p53; increase in FasL level with activation of caspase-8; changes related to members of Bcl-2 family with increase in the level of pro-apoptotic members and decrease in that of anti-apoptotic ones; dissipation of mitochondrial potential with cytochrome c release and activation of caspase-3. In contrast, Chang liver cells exhibited a very low susceptibility to bortezomib-induced apoptosis, which was accompanied by modest modifications in the expression of apoptotic factors. In HepG2 cells bortezomib markedly increased AP-1 activity and the expression of its transcriptional targets such as c-Jun, FasL, BimEL, which are involved in apoptosis. Moreover, AP-1 induced its own production by increasing c-Jun content in the composition of the same AP-1 complex. In addition, bortezomib caused activation of JNK1, which in turn increased the level of phospho-c-Jun as well as stimulated the activation of caspase-3 and t-Bid, two fundamental apoptotic factors. Interestingly, siRNA silencing of c-Jun or JNK1 reduced HepG2 cell susceptibility to apoptosis and prevented the increase in AP-1 activity. Both JNK-1 and AP-1 thus exerted a crucial role in bortezomib-induced apoptosis. Differently, in Chang liver cells the different composition of AP-1 complex as well as the failure of JNK activation seemed to be responsible for the low susceptibility to apoptosis. Given the high susceptibility of hepatoma cells to bortezomib, our results suggest the potential application of this compound in clinical trials for liver cancers.

Butyrate can promote programmed cell death in a number of tumour cells in vitro. This paper provides evidence that butyrate induces apoptosis in human hepatoma HuH-6 and HepG2 cells but is ineffective in Chang liver cells, an immortalised non-tumour cell line. In both HuH-6 and HepG2 cells, apoptosis appeared after a lag period of approximately 16 h and increased rapidly during the second day of treatment. In particular, the effect was stronger in HuH-6 cells, which were, therefore, chosen for ascertaining the mechanism of butyrate action. In HuH-6 cells, beta-catenin seemed to exert an important protective role against apoptosis, since pretreatment with beta-catenin antisense ODN reduced the content of beta-catenin and anticipated the onset of apoptosis at 8 h of exposure to butyrate. Moreover, in HuH-6 cells, butyrate induced loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspase 9 and caspase 3, and degradation of poly(ADP-ribose) polymerase. In addition, during the second day of treatment, beta-catenin, pRb, and cyclins D and E were diminished and the phosphorylated form of pRb disappeared. Also, the content of the anti-apoptotic factor Bcl-XL fell markedly during this period, while that of the pro-apoptotic factor Bcl-Xs increased. These effects were accompanied by an increase in both Bcl-XL and Bcl-Xs mRNA transcripts, as ascertained by reverse transcriptase-polymerase chain reaction. Our results suggest that caspases have a crucial role in butyrate-induced apoptosis. This conclusion is supported by the observation that the inhibitors of caspases, benzyloxy carbonyl-Val-Ala-Asp-fluoromethylketone and benzyloxy carbonyl-Asp-Glu-Val-Asp-fluoromethylketone, prevented apoptosis and the decrease in Bcl-XL, pRb, cyclins and beta-catenin. These effects were most probably responsible for the increased sensitivity of the cells to butyrate-induced apoptosis, which was observed on the second day of treatment.

A potent inhibitor of serine/threonine kinases, staurosporine exerts antiproliferative and apoptotic effects in many cancer cells, although the exact mechanism of its action is still unclear. This study examines the effects of staurosporine on Chang liver cells, an immortalized non-tumor cell line, in comparison with those caused in HuH-6 and HepG2 cells, two human hepatoma cell lines. Our results provide evidence that staurosporine promotes apoptosis in Chang liver cells as observed by flow cytometric analysis and acridine orange/ethidium bromide staining. The effect appeared already after 8 h of treatment and increased with treatment time and dose. After 48 h of exposure to 200 nM staurosporine clear apoptotic signs were observed in about 50% of the cells. Western blotting analysis showed that in Chang liver cells staurosporine induced a marked decrease in the levels of the antiapoptotic factors Bcl-2 (-75%) and Bcl-XL (-50%). Staurosporine also caused loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria and activation of caspase-3. The involvement of caspases in staurosporine-induced cell death was also suggested by the observation that the addition of z-VAD-fmk, a general inhibitor of caspases, suppressed apoptosis. In HuH-6 and HepG2 cells treatment with staurosporine induced the arrest of cells in G2/M phase of cell cycle. This effect was not modified by z-VAD-fmk and was not accompanied by the appearance of biochemical signs of apoptosis. We conclude that staurosporine induced apoptosis in Chang liver cells by a mitochondria-caspase-dependent pathway which was closely correlated with a decrease in Bcl-2 and Bcl-XL levels, while in HuH-6 and HepG2 hepatoma cells the drug caused only an antiproliferative effect.

This report reviews the current status of extensive efforts directed towards the interpretation of crosstalk between apoptosis and proteasome to understanding the molecular mechanism of anticancer agents targeting proteasome, with particular focus on MG132 and PS-341. The discovery that all cancer cells have retained the apoptotic death program has offered to the researchers new biochemical targets to design anticancer drugs. Moreover, the demonstration that proteasome inhibition induces apoptosis and sensitizes cancer cells to traditional tumoricidal agents has proposed the proteasome as an attractive target for development of new anticancer drugs. Since then, a number of both naturally occurring and synthetic inhibitors of the proteasome have been identified. The best characterized and most widely used inhibitors of the proteasome are the peptide aldehydes; among these MG132, due to its broad spectrum of action, low cost and rapid reversibility of action, still remains the first choice to study proteasome function in cell and tissue cultures. Recently, a very potent new class of selective and reversible proteasome inhibitors which contains an inhibitory boronate group has been described. PS-341 represent the first of this promising class of agents that could have application in cancer therapy and it is the only that has progressed to clinical trials.

This report is focused on the apoptotic effect induced by MG132, an inhibitor of 26S proteasome, in human hepatoma HepG2 cells. The results were compared with those obtained with non-transformed human Chang liver cells. MG132 reduced the viability of HepG2 cells in a time- and dose-dependent manner. The effect was in tight connection with the induction of apoptosis, as indicated by fluorescence microscopy and cytometric analysis, and was accompanied by a remarkable increase in the production of H2O2 and a reduction in mitochondrial transmembrane potential (Deltapsim). In addition cell death was prevented by antioxidants such as GSH, N-acetylcysteine or catalase. Western blot analysis showed that HepG2 cells contain a very low level of Bcl-2 and a much higher level of Bcl-XL, another antiapoptotic factor of the same family. When the cells were exposed to MG132 the level of Bcl-XL diminished, while a new band, corresponding to the expression of the proapoptotic protein Bcl-XS was detected. MG132 also caused the release of cytochrome c from mitochondria and the activation of caspase-3 with the consequent degradation of poly-ADP ribose polymerase (PARP). The observation that the broad spectrum caspase inhibitor z-VAD markedly reduced the apoptotic effect of the drug clearly demonstrated that caspases play an important role in MG132-induced apoptosis. MG132 exerted a modest effect on the viability of Chang liver cells which primarily depended on the G2/M arrest of cell cycle while only a small percentage of apoptotic cells was found. The remarkable differences in the effects induced by MG132 in Chang liver cells and HepG2 cells made us hypothesise the potential use of proteasome inhibitors in hepatocarcinoma therapy.

This paper studies the cytotoxic effect induced by the topoisomerase I inhibitor camptothecin in human osteosarcoma Saos-2 cells, which lack p53 and contain a non-functional form of the product of the retinoblastoma gene, pRb. Cytotoxicity induced by camptothecin was dose- and time-dependent; the treatment with 100 nM camptothecin reduced cell viability by 50% at 32 h and by 75% at 72 h of exposure. The cytotoxic effect was caused by apoptosis, as ascertained by morphological evidence, acridine orange-ethidium bromide staining and flow cytometric analysis. Apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase. Treatment with camptothecin caused a threefold increase in the activity of c-Jun N-terminal kinase (JNK) and an eightfold increase in the level of phosphorylated c-Jun. The introduction of the RB gene into Saos-2 cells reduced the rate of cell growth. Moreover, stable clones of transfected cells were resistant to camptothecin. Exposure to 100 nM camptothecin for 72 h reduced the viability of transfected cells by only 10%; moreover, very modest effects were observed on the activity of JNK as well as on the level of phosphorylated c-Jun. The results reported in this paper support the conclusion that the expression of wild-type pRb in Saos-2 cells exerts an anti-apoptotic influence through the control of JNK activity.

Our results demonstrate that sodium phenylbutyrate, a compound with a low degree of toxicity, exerted a cytotoxic effect on human retinoblastoma Y79 cells in a time- and dose-dependent manner. Treatment of Y79 cells for 72 h with phenylbutyrate reduced cell viability by 63% at 2 mM and 90% at 4 mM. Cell death caused by phenylbutyrate exhibited the typical features of apoptosis, as shown by light and fluorescent microscopy. Western blot analysis demonstrated that exposure of Y79 cells to phenylbutyrate decreased the level of the antiapoptotic factor Bcl-2 and induced the activation of caspase-3, a key enzyme in the execution phase of apoptosis. Moreover, treatment with phenylbutyrate markedly increased the level of acetylated histone-H3. Combined treatment with phenylbutyrate and topotecan, a topoisomerase I-inhibitor, resulted in a clear synergistic effect. We suggest that the effects exerted by phenylbutyrate on Y79 cells essentially depend on modifications of gene expression consequent to histone hyperacetylation.

Methyl gallate (MG), which is a gallotannin widely found in plants, is a polyphenol used in traditional Chinese phytotherapy to alleviate several cancer symptoms. Our studies provided evidence that MG is capable of reducing the viability of HCT116 colon cancer cells, while it was found to be ineffective on differentiated Caco-2 cells, which is a model of polarized colon cells. In the first phase of treatment, MG promoted both early ROS generation and endoplasmic reticulum (ER) stress, sustained by elevated PERK, Grp78 and CHOP expression levels, as well as an upregulation in intracellular calcium content. Such events were accompanied by an autophagic process (16–24 h), where prolonging the time (48 h) of MG exposure led to cellular homeostasis collapse and apoptotic cell death with DNA fragmentation and p53 and γH2Ax activation. Our data demonstrated that a crucial role in the MG-induced mechanism is played by p53. Its level, which increased precociously (4 h) in MG-treated cells, was tightly intertwined with oxidative injury. Indeed, the addition of N-acetylcysteine (NAC), which is a ROS scavenger, counteracted the p53 increase, as well as the MG effect on cell viability. Moreover, MG promoted p53 accumulation into the nucleus and its inhibition by pifithrin-α (PFT-α), which is a negative modulator of p53 transcriptional activity, enhanced autophagy, increased the LC3-II level and inhibited apoptotic cell death. These findings provide new clues to the potential action of MG as a possible anti-tumor phytomolecule for colon cancer treatment.

Oncogenic BRAF mutations have been widely described in melanomas and promote tumour progression and chemoresistance. We previously provided evidence that the HDAC inhibitor ITF2357 (Givinostat) targets oncogenic BRAF in SK-MEL-28 and A375 melanoma cells. Here, we show that oncogenic BRAF localises to the nucleus of these cells, and the compound decreases BRAF levels in both the nuclear and cytosolic compartments. Although mutations in the tumour suppressor p53 gene are not equally frequent in melanomas compared to BRAF, the functional impairment of the p53 pathway may also contribute to melanoma development and aggressiveness. To understand whether oncogenic BRAF and p53 may cooperate, a possible interplay was considered in the two cell lines displaying a different p53 status, being p53 mutated into an oncogenic form in SK-MEL-28 and wild-type in A375 cells. Immunoprecipitation revealed that BRAF seems to preferentially interact with oncogenic p53. Interestingly, ITF2357 not only reduced BRAF levels but also oncogenic p53 levels in SK-MEL-28 cells. ITF2357 also targeted BRAF in A375 cells but not wild-type p53, which increased, most likely favouring apoptosis. Silencing experiments confirmed that the response to ITF2357 in BRAF-mutated cells depends on p53 status, thus providing a rationale for melanoma-targeted therapy.

Histone deacetylase inhibitors (HDACI) are epigenetic compounds that have been widely considered very promising antitumor agents. Here, we focus on the effects of the pan-HDAC inhibitor ITF2357 (Givinostat) in comparison with SAHA (Vorinostat) in melanoma cells bearing BRAF V600E oncogenic mutation. Our results indicate both ITF2357 and SAHA dose-dependently reduce the viability of BRAF-mutated SK-MEL-28 and A375 melanoma cells. The comparison of IC50 values revealed that ITF2357 was much more effective than SAHA. Interestingly, both inhibitors markedly decreased oncogenic BRAF protein expression levels, ITF2357 being the most effective compound. Moreover, the BRAF decrease induced by ITF2357 was accompanied by a decrease in the level of phospho-ERK1/2. The inhibitor of upstream MEK activity, U0126, reduced ERK1/2 phosphorylation and dramatically potentiated the antitumor effect of ITF2357, exacerbating the reduction in the BRAF level. ITF2357 stimulated an early pro-survival autophagic response, which was followed by apoptosis, as indicated by apoptotic markers evaluation and the protective effects exerted by the pan-caspase inhibitor z-VADfmk. Overall, our data indicate for the first time that ITF2357 targets oncogenic BRAF in melanoma cells and induces a switch from autophagy to classic apoptosis, thus representing a possible candidate in melanoma targeted therapy.

Due to its chemical properties and multiple molecular effects on different tumor cell types, the sesquiterpene lactone parthenolide (PN) can be considered an effective drug with significant potential in cancer therapy. PN has been shown to induce either classic apoptosis or alternative caspase-independent forms of cell death in many tumor models. The therapeutical potential of PN has been increased by chemical design and synthesis of more soluble analogues including dimethylaminoparthenolide (DMAPT). This review focuses on the molecular mechanisms of both PN and analogues action in tumor models, highlighting their effects on gene expression, signal transduction and execution of different types of cell death. Recent findings indicate that these compounds not only inhibit prosurvival transcriptional factors such as NF-κB and STATs but can also determine the activation of specific death pathways, increasing intracellular reactive oxygen species (ROS) production and modifications of Bcl-2 family members. An intriguing property of these compounds is its specific targeting of cancer stem cells. The unusual actions of PN and its analogues make these agents good candidates for molecular targeted cancer therapy.

p62 is a versatile protein involved in the delicate balance between cell death and survival, which is fundamental for cell fate decision in the context of both cancer and neurodegenerative diseases. As an autophagy adaptor, p62 recognizes polyubiquitin chains and interacts with LC3, thereby targeting the selected cargo to the autophagosome with consequent autophagic degradation. Beside this function, p62 behaves as an interactive hub in multiple signalling including those mediated by Nrf2, NF-κB, caspase-8, and mTORC1. The protein is thus crucial for the control of oxidative stress, inflammation and cell survival, apoptosis, and metabolic reprogramming, respectively. As a multifunctional protein, p62 falls into the category of those factors that can exert opposite roles in the cells. Chronic p62 accumulation was found in many types of tumors as well as in stress granules present in different forms of neurodegenerative diseases. However, the protein seems to have a Janus behaviour since it may also serve protective functions against tumorigenesis or neurodegeneration. This review describes the diversified roles of p62 through its multiple domains and interactors and specifically focuses on its oncoJanus and neuroJanus roles.

Oncogenic BRAF mutations have been widely described in melanomas and promote tumour progression and chemoresistance. We previously provided evidence that the HDAC inhibitor ITF2357 (Givinostat) targets oncogenic BRAF in SK-MEL-28 and A375 melanoma cells. Here, we show that oncogenic BRAF localises to the nucleus of these cells, and the compound decreases BRAF levels in both the nuclear and cytosolic compartments. Although mutations in the tumour suppressor p53 gene are not equally frequent in melanomas compared to BRAF, the functional impairment of the p53 pathway may also contribute to melanoma development and aggressiveness. To understand whether oncogenic BRAF and p53 may cooperate, a possible interplay was considered in the two cell lines displaying a different p53 status, being p53 mutated into an oncogenic form in SK-MEL-28 and wild-type in A375 cells. Immunoprecipitation revealed that BRAF seems to preferentially interact with oncogenic p53. Interestingly, ITF2357 not only reduced BRAF levels but also oncogenic p53 levels in SK-MEL-28 cells. ITF2357 also targeted BRAF in A375 cells but not wild-type p53, which increased, most likely favouring apoptosis. Silencing experiments confirmed that the response to ITF2357 in BRAF-mutated cells depends on p53 status, thus providing a rationale for melanoma-targeted therapy. [ABSTRACT FROM AUTHOR]

Methyl gallate (MG), which is a gallotannin widely found in plants, is a polyphenol used in traditional Chinese phytotherapy to alleviate several cancer symptoms. Our studies provided evidence that MG is capable of reducing the viability of HCT116 colon cancer cells, while it was found to be ineffective on differentiated Caco-2 cells, which is a model of polarized colon cells. In the first phase of treatment, MG promoted both early ROS generation and endoplasmic reticulum (ER) stress, sustained by elevated PERK, Grp78 and CHOP expression levels, as well as an upregulation in intracellular calcium content. Such events were accompanied by an autophagic process (16–24 h), where prolonging the time (48 h) of MG exposure led to cellular homeostasis collapse and apoptotic cell death with DNA fragmentation and p53 and γH2Ax activation. Our data demonstrated that a crucial role in the MG-induced mechanism is played by p53. Its level, which increased precociously (4 h) in MG-treated cells, was tightly intertwined with oxidative injury. Indeed, the addition of N-acetylcysteine (NAC), which is a ROS scavenger, counteracted the p53 increase, as well as the MG effect on cell viability. Moreover, MG promoted p53 accumulation into the nucleus and its inhibition by pifithrin-α (PFT-α), which is a negative modulator of p53 transcriptional activity, enhanced autophagy, increased the LC3-II level and inhibited apoptotic cell death. These findings provide new clues to the potential action of MG as a possible anti-tumor phytomolecule for colon cancer treatment. [ABSTRACT FROM AUTHOR]

Ethanolic extracts from Mangifera indica L. have been proved to possess anti-tumor properties in many cancer systems. However, although most effects have been demonstrated with fruit pulp extract, the underlying molecular mechanisms of mango peel are still unclear. This study was designed to explore the effects of mango peel extract (MPE) on colon cancer cell lines. MPE affected cell viability and inhibited the colony formation trend of tumor cells, while no effects were observed in human dermal fibroblasts used as a non-cancerous cell line model. These events were a consequence of the induction of apoptosis associated to reactive oxygen species (ROS) production, activation of players of the oxidative response such as JNK and ERK1/2, and the increase in Nrf2 and manganese superoxide dismutase (MnSOD). Significantly, mango peel-activated stress triggered a DNA damage response evidenced by the precocious phosphorylation of histone 2AX (γH2AX), as well as phosphorylated Ataxia telangiectasia-mutated (ATM) kinase and p53 upregulation. Mango peel extract was also characterized, and HPLC/MS (High Performance Liquid Chromatography/Mass Spectrometry) analysis unveiled the presence of some phenolic compounds that could be responsible for the anti-cancer effects. Collectively, these findings point out the importance of the genotoxic stress signaling pathway mediated by γH2AX in targeting colon tumor cells to apoptosis.

Histone deacetylase inhibitors (HDACI) are epigenetic compounds that have been widely considered very promising antitumor agents. Here, we focus on the effects of the pan-HDAC inhibitor ITF2357 (Givinostat) in comparison with SAHA (Vorinostat) in melanoma cells bearing BRAF V600E oncogenic mutation. Our results indicate both ITF2357 and SAHA dose-dependently reduce the viability of BRAF-mutated SK-MEL-28 and A375 melanoma cells. The comparison of IC50 values revealed that ITF2357 was much more effective than SAHA. Interestingly, both inhibitors markedly decreased oncogenic BRAF protein expression levels, ITF2357 being the most effective compound. Moreover, the BRAF decrease induced by ITF2357 was accompanied by a decrease in the level of phospho-ERK1/2. The inhibitor of upstream MEK activity, U0126, reduced ERK1/2 phosphorylation and dramatically potentiated the antitumor effect of ITF2357, exacerbating the reduction in the BRAF level. ITF2357 stimulated an early pro-survival autophagic response, which was followed by apoptosis, as indicated by apoptotic markers evaluation and the protective effects exerted by the pan-caspase inhibitor z-VADfmk. Overall, our data indicate for the first time that ITF2357 targets oncogenic BRAF in melanoma cells and induces a switch from autophagy to classic apoptosis, thus representing a possible candidate in melanoma targeted therapy. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A series of new 4-acetamidobenzamides bearing the pyrazole or indazole nucleus was synthesized. • Synthesized compounds were evaluated for the antiproliferative activity against human lung carcinoma H292 cells. • They caused growth inhibition at micromolar doses. • The mechanism of action was studied. Abstract Based on some common structural features of known compounds interfering with p53 pathways and our previously synthesized benzamides, we synthesized new ethyl 5-(4-substituted benzamido)-1-phenyl-1H-pyrazole-4-carboxylates 26a-c , ethyl 5-(4-substituted benzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carboxylates 27a-c and N-(1H-indazol-6-yl)-4-substituted benzamides 31a,b bearing in the 4 position of the benzamido moiety the 2-phenylpropanamido or 2-phenoxyacetamido or cinnamamido groups. A preliminary test to evaluate the antiproliferative activity against human lung carcinoma H292 cells highlighted how compound 26c showed the best activity. This last was therefore selected for further studies with the aim to find the mechanism of action. Compound 26c induces intrinsic apoptotic pathway by activating p53 and is also able to activate TRAIL-inducing death pathway by promoting increase of DR4 and DR5 death receptors, downregulation of c-FLIP L and caspase-8 activation. [ABSTRACT FROM AUTHOR]