Sulforaphane impaired immune checkpoint blockade therapy through activating ΔNP63α/PD-L1 axis in gastric cancer.

Sulforaphane (SFN) exerts anticancer effect on various cancers including gastric cancer. However, the regulatory effect of SFN on programmed death-ligand 1 (PD-L1) and checkpoint blockade therapy in gastric cancer have not been elucidated. Here we demonstrated that SFN suppressed gastric cancer cell growth both in vitro and in vivo study. SFN upregulated PD-L1 expression through activating ΔNP63α in gastric cancer cells. Further, we found that SFN impaired the anticancer effect of anti-PD-L1 monoclonal antibody (α-PD-L1 mab) on gastric cancer cells. These results uncover a novel PD-L1 regulatory mechanism and the double-edged role of SFN in gastric cancer intervention.

Diallyl trisulfide induces pyroptosis and impairs lung CSC-like properties by activating the ROS/Caspase 1 signaling pathway.

Lung cancer stem cells (CSCs) drive continuous cancer growth and metastatic dissemination; thus, there is an urgent requirement to acquire effective therapeutic strategies for targeting lung CSCs. Diallyl trisulfide (DATS), a garlic organosulfide, possesses suppressive potential in lung cancer; however, its underlying mechanism is still unclear. In this study, we identified DATS as a pyroptosis inducer in lung cancer cells. DATS-treated A549 and H460 cells exhibited pyroptotic cell death, with characteristic large bubbles appearing on their plasma membrane and LDH release. DATS induced cell death, arrested the cell cycle at the G2/M phase, and inhibited colony formation in lung cancer cells. Meanwhile, we found that DATS significantly suppressed the malignant features by impairing lung CSC-like properties, including sphere formation ability, CD133 positive cell number, and lung CSCs marker expression. Mechanistically, DATS induced cell pyroptosis via increasing the expression of NLRP3, ASC, Pro Caspase 1, Cleaved Caspase 1, GSDMD, GSDMD-N, and IL-1ß. The verification experiments showed that the effects of DATS on pyroptosis and lung CSC-like properties were weakened after Caspase 1 inhibitor VX-765 treatment, indicating that DATS activated NLRP3 inflammasome-mediated pyroptosis by targeting Caspase 1 in lung cancer cells. Moreover, DATS increased ROS overproduction and mitochondrial dysfunction, which contributed to DATS-induced pyroptosis of lung cancer cells. NAC treatment reversed the effects of DATS on pyroptosis and CSC-like properties. In vivo experiment further confirmed that DATS restrained tumor growth. Together, our results suggest that DATS promotes pyroptosis and impairs lung CSC-like properties by activating ROS/Caspase 1 signaling pathway, thereby retarding lung cancer progression.

Phytochemicals in regulating PD-1/PD-L1 and immune checkpoint blockade therapy.

Clinical treatment and preclinical studies have highlighted the role of immune checkpoint blockade in cancer treatment. Research has been devoted to developing immune checkpoint inhibitors in combination with other drugs to achieve better efficacy or reduce adverse effects. Phytochemicals sourced from vegetables and fruits have demonstrated antiproliferative, proapoptotic, anti-migratory, and antiangiogenic effects against several cancers. Phytochemicals also modulate the tumor microenvironment such as T cells, regulatory T cells, and cytokines. Recently, several phytochemicals have been reported to modulate immune checkpoint proteins in in vivo or in vitro models. Phytochemicals decreased programmed cell death ligand-1 expression and synergized programmed cell death receptor 1 (PD-1) monoclonal antibody to suppress tumor growth. Combined administration of phytochemicals and PD-1 monoclonal antibody enhanced the tumor growth inhibition as well as CD4+ /CD8+ T-cell infiltration. In this review, we discuss immune checkpoint molecules as potential therapeutic targets of cancers. We further assess the impact of phytochemicals including carotenoids, polyphenols, saponins, and organosulfur compounds on cancer PD-1/programmed cell death ligand-1 immune checkpoint molecules and document their combination effects with immune checkpoint inhibitors on various malignancies.

ZNF217-activated Notch signaling mediates sulforaphane-suppressed stem cell properties in colorectal cancer.

Cancer stem cells (CSCs) are known to contribute to the progression of colorectal cancer (CRC). However, understanding of the molecular mechanisms and key factors involved in CRC is still insufficient to identify therapeutic targets against colorectal CSCs. In an effort to identify such mechanisms, we conducted bioinformatics analyses to evaluate the expression patterns in tumor and normal colorectal tissues, leading us to focus on the role of the ZNF217/Notch1 axis in mediating stem cell properties in CRC. Our findings revealed that ZNF217 overexpression activated self-renewal ability, expression of colorectal CSC markers, and Notch signaling in CRC. Dual-luciferase reporter assay suggested a role for ZNF217 in targeting Notch1 to activate Notch signaling. We observed that the promotional effects of Notch signaling, as well as CSC markers, under ZNF217 overexpression were attenuated after Notch1 knockdown. In addition to in vitro data, our in vivo results confirmed the inhibitory effect of sulforaphane on the tumorigenicity of CSCs, depicted the suppressive role of sulforaphane on colorectal CSCs mediated by the ZNF217/Notch1 axis, thereby providing new targetable vulnerabilities and therapeutic strategies for CRC.

Diallyl trisulfide inhibits gastric cancer stem cell properties through ΔNp63/sonic hedgehog pathway.

Gastric cancer is one of the deadliest malignant tumors, and half of the patients develop recurrences or metastasis within 5 years after eradication therapy. Cancer stem cells (CSCs) are considered to be important in this progress. The sonic hedgehog (SHH) pathway plays an important role in the maintenance of gastric CSCs characteristics. The p63 proteins are vital transcription factors belonging to the p53 family, while their functions in regulating CSCs remain unclear. The preventive effects of dietary diallyl trisulfide (DATS) against human gastric cancer have been verified. However, whether DATS can target gastric CSCs are poorly understood. Here, we investigated the role of ΔNp63/SHH pathway in gastric CSCs and the inhibitory effect of DATS on gastric CSCs via ΔNp63/SHH pathway. We found that ΔNp63 was upregulated in serum-free medium cultured gastric tumorspheres compared with the parental cells. Overexpression of ΔNp63 elevated the self-renewal capacity and CSC markers' levels in gastric sphere-forming cells. Furthermore, we found that ΔNp63 directly bound to the promoter region of Gli1, the key transcriptional factor of SHH pathway, to enhance its expression and to activate SHH pathway. In addition, it was revealed that DATS effectively inhibited gastric CSC properties both in vitro and in vivo settings. Activation of SHH pathway attenuated the suppressive effects of DATS on the stemness of gastric cancer. Moreover, DATS suppression of gastric CSC properties was also diminished by ΔNp63 upregulation through SHH pathway activation. These findings illustrated the role of ΔNp63/SHH pathway in DATS inhibition of gastric cancer stemness. Taken together, the present study suggested for the first time that DATS inhibited gastric CSCs properties by ΔNp63/SHH pathway.

[Corrigendum] Curcumin reverses benzidine-induced epithelial-mesenchymal transition via suppression of ERK5/AP-1 in SV-40 immortalized human urothelial cells.

Following the publication of the above article, an interested reader drew to the authors' attention that, for the Transwell invasion assays shown in Fig. 5D on p. 1326, the images selected for the '0 µM benzidine / 0 µM curcumin' and '0 µM benzidine / 1 µM curcumin' experiments were overlapping, such that these data appeared to have been derived from the same original source. After having consulted their original data, the authors have realized that the '0 µM benzidine / 1 µM curcumin' data panel was selected incorrectly. The revised version of Fig. 5, showing the correct data for the '0 µM benzidine / 1 µM curcumin' data panel in Fig. 5D, is shown on the next page. The authors regret that this error went unnoticed prior to the publication of this article, and thank the Editor of International Journal of Oncology for allowing them the opportunity to publish this corrigendum. All the authors agree with the publication of this corrigendum; furthermore, they also apologize to the readership of the journal for any inconvenience caused.[International Journal of Oncology 50: 1321­1329, 2017; DOI: 10.3892/ijo.2017.3887].

Destruction of the cellular antioxidant pool contributes to resveratrol-induced senescence and apoptosis in lung cancer.

Resveratrol (RES) has various pharmacological bioactivities and its anticancer effects in lung cancer have been proven. However, the underlying mechanisms of action of RES in lung cancer remain unclear. This study focused on Nrf2-mediated antioxidant systems in RES-treated lung cancer cells. A549 and H1299 cells were treated with various concentrations of RES at different times. RES decreased cell viability, inhibited cell proliferation, and increased the number of senescent and apoptotic cells in a concentration- and time-dependent manner. Moreover, RES-induced lung cancer cell arrest at the G1 phase was accompanied by changes in apoptotic proteins (Bax, Bcl-2, and cleaved caspase 3). Furthermore, RES induced a senescent phenotype along with changes in senescence-related markers (senescence-associated ß-galactosidase activity, p21, and p-γH2AX). More importantly, with prolonged exposure time and increased exposure concentration, intracellular reactive oxygen species (ROS) continuously accumulated, resulting in a decrease in Nrf2 and its downstream antioxidant response elements, including CAT, HO-1, NQO1, and SOD1. Meanwhile, RES-induced ROS accumulation and cell apoptosis were reversed by N-acetyl-l-cysteine treatment. Taken together, these results suggest that RES disturb lung cancer cellular homeostasis by destroying the intracellular antioxidant pool to increase ROS production. Our findings provide a new perspective on RES intervention in lung cancer.

Low-dose phthalates promote breast cancer stem cell properties via the oncogene ΔNp63α and the Sonic hedgehog pathway.

BACKGROUND: The omnipresence of human phthalate (PAE) exposure is linked to various adverse health issues, including breast cancer. However, the effects of low-dose PAE exposure on breast cancer stem cells (BCSCs) and the underlying mechanism remain unexplored. METHODS: BCSCs from breast cancer cell lines (MDA-MB-231 and MCF-7) were enriched using a tumorsphere formation assay. Gene and protein expression was detected by measurement of quantitative real-time reverse transcription PCR, western blot, and immunofluorescence assays. Transient transfection assays were used to evaluate the involvement of Gli1, a signaling pathway molecule and ΔNp63α, an oncogene in influencing the PAE-induced characteristics of BCSCs. RESULTS: PAE (butylbenzyl phthalate, BBP; di-butyl phthalate, DBP; di-2-ethylhexyl phthalate, DEHP) exposure of 10-9 M significantly promoted the tumorsphere formation ability in BCSCs. Breast cancer spheroids with a 10-9 M PAE exposure had higher levels of BCSC marker mRNA and protein expression, activated sonic hedgehog (SHH) pathway, and increased mRNA and protein levels of an oncogene, ΔNp63α. Furthermore, suppression of the SHH pathway attenuated the effects of PAEs on BCSCs. And the overexpression of ΔNp63α enhanced PAE-induced characteristics of BCSCs, while low expression of ΔNp63α inhibited the promotion effects of PAEs on BCSCs and the SHH pathway. CONCLUSION: Low-dose PAE exposure promoted the stem cell properties of BCSCs in a ΔNp63α- and SHH-dependent manner. The influence of low-dose exposure of PAEs and its relevance for the lowest observed effect concentrations requires further investigation, and the precise underlying mechanism needs to be further explored.

Sulforaphane ameliorates bisphenol A-induced hepatic lipid accumulation by inhibiting endoplasmic reticulum stress.

The aim of the present study was to investigate the role of endoplasmic reticulum (ER) stress in bisphenol A (BPA) - induced hepatic lipid accumulation as well as the protective effects of Sulforaphane (SFN) in this process. Human hepatocyte cell line (LO2) and C57/BL6J mice were used to examine BPA-triggered hepatic lipid accumulation and the underlying mechanism. Hepatic lipid accumulation, triglycerides (TGs) levels, the expression levels of lipogenesis-related genes and proteins in the ER stress pathway were measured. It was revealed that BPA treatment increased the number of lipid droplets, the levels of TG and mRNAs expression of lipogenesis-related genes, and activated the ER stress pathway. These changes were inhibited by an ER stress inhibitor 4-phenylbutyric acid. SFN treatment abrogated BPA-altered hepatic lipid metabolism and ameliorated BPA-induced ER stress-related markers. Together, these findings suggested that BPA activated ER stress to promote hepatic lipid accumulation, and that SFN reversed those BPA effects by alleviating ER stress.

Sulforaphane Suppresses MCF-7 Breast Cancer Cells Growth via miR-19/PTEN Axis to Antagonize the Effect of Butyl Benzyl Phthalate.

Sulforaphane (SFN), a major isothiocyanate found in cruciferous vegetables, reportedly exerts extensive antitumor effects. Butyl benzyl phthalate (BBP), a widely used plasticizer, plays a crucial role in the promotion of breast cancer. In the present study, we demonstrated that SFN inhibited proliferation, induced apoptosis, and suppressed the stemness of MCF-7 cells, whereas BBP exerted the opposite effects; microRNA-19 (miR-19) plays an important role in BBP-induced cell growth and dysregulation mediated via PTEN and p21. The growth-promoting effect of BBP could be mitigated by SFN, accompanied by a reversal of altered expression of miR-19a, miR-19b, PTEN, and p21. SFN also suppressed BBP-induced binding of upregulated miR-19 with PTEN, as determined using a dual-luciferase reporter assay. Collectively, these results demonstrated, for the first time, that SFN regulates the miR-19/PTEN axis to exert protective effects against BBP-mediated breast cancer promotion, suggesting a new potential role for SFN (or SFN-rich foods) in phthalate antagonism.

Isothiocyanates attenuate immune checkpoint blockage therapy in gastric cancer via induction of PD-L1 expression.

The PD-1/PD-L1 immune checkpoint blockade therapy has shown revolutionary efficacy in the treatment of multiple cancers including gastric cancer. Isothiocyanates play important roles in cancer cell suppression and immunomodulation. However, the effects of isothiocyanates on immune checkpoint inhibitors are poorly understood in gastric cancer. The influence of three major isothiocyanates (sulforaphane, phenylethyl isothiocyanate, and benzhydryl isothiocyanate) on gastric cancer cell growth and PD-L1 expression was investigated. Syngeneic mouse models were administered by isothiocyanates and anti-PD-L1 monoclonal antibody, and the anti-tumor effects were assessed. The expression of PD-L1, proportion of lymphocytes and serum cytokine levels were detected to explore the underlying mechanisms. We found that PD-L1 expression was significantly induced by isothiocyanates which was associated with TAp63α up-regulation. We further revealed that TAp63α promoted PD-L1 through transcriptional activation. Combination treatment of isothiocyanates and anti-PD-L1 therapy weakened the sensitivity of gastric cancer cells to anti-PD-L1 drug. Moreover, in vivo studies illustrated that the interference effects of isothiocyanates on anti-PD-L1 antibody were related to PD-L1 expression and decreased infiltrating T lymphocytes in tumor bearing mouse hosts. Our findings provide novel insights as isothiocyanates could interfere with the successful application of immunotherapy in gastric cancer.

Diallyl Trisulfide Suppresses the Renal Cancer Stem-like Cell Properties via Nanog.

Cancer stem-like cells (CSCs), which play an important role in tumor initiation and progression, have been identified in many cancers. Diallyl trisulfide (DATS) is an organosulfur compound extracted from garlic with anticancer activities. Nanog is a transcription factor responsible for maintaining the stemness of CSCs, but its role in the DATS-induced attenuation of renal CSC properties is unknown. In this study, renal CSCs were enriched from human renal cancer cell lines 786-O and ACHN cultured in a serum-free medium (SFM). The properties of CSCs were analyzed by evaluating the ability of the cells in sphere formation and measuring the expression of stem cell markers. We found that downregulation of Nanog inhibited renal CSC properties. DATS suppressed renal CSC activities by reducing tumorsphere formation, decreasing stem cell markers including Nanog, CD44, ALDH1A1, and Oct4, inhibiting cell proliferation and promoting apoptosis. We further revealed that overexpression of Nanog reversed the suppressive effects of DATS on renal CSCs. Taken together, our results demonstrated that DATS inhibited renal CSCs by suppressing Nanog. These novel findings suggested that, through Nanog targeting, DATS can potentially be used as an anti-tumor agent for renal cancer.

Resveratrol suppresses lung cancer by targeting cancer stem-like cells and regulating tumor microenvironment.

Increasing evidence indicate that cancer stem cells (CSCs) are the key driver of tumor initiation and recurrence. The cellular and soluble components of the tumor microenvironment (TME) impact on cancer initiation and progression, such as cytokines and chemokines. Thus, targeting CSCs and TME is a novel anti-cancer approach. Resveratrol (RES), a bioactive phytochemical extracted from various plants, exhibits tumor-suppressing activities in lung cancer, yet the mechanism remains poorly understood. Our data showed that the expression level of IL-6 was positively correlated with the presence of lung cancer stem-like cells (LCSCs) in human lung cancer tissues. In vitro results showed that IL-6 was highly elevated in lung cancer sphere-forming cells and could enhance the stemness of LCSCs, including tumor sphere formation ability, the percentage of CD133 positive cells, and the expression of LCSC specific markers (CD133, ALDH1A1 and Nanog). Simultaneously, our results confirmed that RES effectively inhibited LCSC properties, downregulated Wnt/ß-catenin signaling and reduced IL-6 level in vitro and in vivo. Furthermore, we found RES treatment attenuated the activation of Wnt/ß-catenin signaling by LiCl (GSK3ß agonist). IL-6-promoted LCSC properties and Wnt/ß-catenin signaling was also reversed by RES. Taken together, these data illustrated that RES inhibited lung cancer by targeting LCSCs and IL-6 in TME. The novel findings from this study provided evidence that RES exhibited multi-target effects on suppression of lung cancer and could be a novel potent cancer-preventive compound.

Regulation of ZO-1 on ß-catenin mediates sulforaphane suppressed colorectal cancer stem cell properties in colorectal cancer.

Cancer stem cells (CSCs) function as the driving force of cancer initiation and progression. Wnt/ß-catenin is the core pluripotency pathway in CSCs, while its crucial regulator has not been fully elucidated yet. Here, we evaluated the role of ZO-1, a component of the tight junction protein complex, in colorectal CSCs, and found ZO-1 downregulation in both colorectal cancer cells and spheres. Over-expression of ZO-1 can inhibit the sphere-forming capacity and CSC marker expression in spheres. Immunofluorescence staining and co-immunoprecipitation analysis further revealed the interaction between ZO-1 and ß-catenin and the repressed role of ZO-1 in ß-catenin nuclear accumulation. Using in vitro and in vivo models, we suggested the suppression effects of sulforaphane on CSCs via the ZO-1/ß-catenin axis in colorectal cancer. The findings from this study depicted for the first time that ZO-1 dampened colorectal CSCs by interacting with ß-catenin and attenuated its nuclear translocation, providing new insights into the mechanisms and applications of sulforaphane in targeting CSCs.

Magnesium isoglycyrrhizinate ameliorates lipopolysaccharide-induced liver injury by upregulating autophagy and inhibiting inflammation via IL-22 expression.

Liver disease has become a major cause of premature mortality worldwide. It is well known that dysregulated inflammation response plays a crucial role in most liver diseases. As a Chinese medicinal herb, Magnesium isoglycyrrhizinate (MgIG) has been proven to have good hepatoprotective activity and has been used in clinic to treat liver disease. However, the mechanisms by which MgIG regulates LPS-induced liver injury and inflammation in vivo remain elusive. In our study, MgIG pretreatment mitigated LPS-induced liver damage by suppressing apoptosis and inflammation via regulating macrophage/neutrophil infiltration. MgIG ameliorated the effects of LPS on pro-oxidant enzymes (NOX1/2/4) and anti-oxidant enzymes (SOD1/2). Interestingly, we found that the level of the hepatoprotective cytokine interleukin (IL)-22 was significantly upregulated in MgIG-treated liver tissues, which might be a potential mechanism of MgIG against liver injury. Moreover, we found that MgIG treatment not only inhibited TLR4/MyD88/NF-κB signaling pathway, but also activated autophagy. Furthermore, IL-22 treatment activated autophagy and inhibited TLR4/NF-κB signaling pathway in vitro, suggesting that IL-22-activated autophagy and -inhibited inflammation also participated in the protective effects of MgIG. Altogether, our results uncovered the potential mechanisms of the hepatoprotective effects of MgIG, which provided critical evidence to support the use of MgIG to prevent and treat liver diseases.

ΔNp63α mediates sulforaphane suppressed colorectal cancer stem cell properties through transcriptional regulation of Nanog/Oct4/Sox2.

Cancer stem cells (CSCs) play a key role in cancer initiation, development, metastasis, and recurrence. Previously, we found that sulforaphane (SFN), a natural compound obtained from cruciferous vegetables, inhibited colorectal CSCs via the downregulation of TAp63α. However, the role of ΔNp63α, another critical isoform of p63 which has been considered to contribute to cancer progression, in SFN-mediated colorectal CSCs inhibition remains unclear. Here, we showed that ΔNp63α expression was enhanced in sphere-forming colorectal cancer cells. Overexpression of ΔNp63α promoted the properties of CSCs, while downregulation of ΔNp63α suppressed those properties. Besides, ΔNp63α was found to activate the transcription of core CSCs genes including Nanog, Oct4, and Sox2. Furthermore, in vitro and in vivo experiments illustrated the regulatory effects of SFN on ΔNp63α and colorectal CSCs. These findings suggested for the first time that ΔNp63α activated the transcription of Nanog, Oct4, Sox2 and mediated the interventional effects of SFN on colorectal CSCs, thus providing a novel mechanism by which SFN inhibits colorectal CSCs.

Interleukin-17A mediates tobacco smoke-induced lung cancer epithelial-mesenchymal transition through transcriptional regulation of ΔNp63α on miR-19.

Interleukin-17A (IL-17A) is an essential inflammatory cytokine in the progress of carcinogenesis. Tobacco smoke (TS) is a major risk factor of lung cancer that influences epithelial-mesenchymal transition (EMT) process. However, the potential mechanism by which IL-17A mediates the progression of lung cancer in TS-induced EMT remains elusive. In the present study, it was revealed that the IL-17A level was elevated in lung cancer tissues, especially in tumor tissues of cases with experience of smoking, and a higher IL-17A level was correlated with induction of EMT in those specimens. Moreover, the expression of ΔNp63α was increased in IL-17A-stimulated lung cancer cells. ΔNp63α functioned as a key oncogene that bound to the miR-17-92 cluster promoter and transcriptionally increased the expression of miR-19 in lung cancer cells. Overexpression of miR-19 promoted EMT in lung cancer with downregulation of E-cadherin and upregulation of N-cadherin, while its inhibition suppressed EMT. Finally, the upregulated levels of IL-17A, ΔNp63α, and miR-19 along with the alteration of EMT-associated biomarkers were found in lung tissues of TS-exposed mice. Taken together, the abovementioned results suggest that IL-17A increases ΔNp63α expression, transcriptionally elevates miR-19 expression, and promotes TS-induced EMT in lung cancer. These findings may provide a new insight for the identification of therapeutic targets for lung cancer.