5'-Nitro-indirubinoxime, an indirubin derivative, suppresses metastatic ability of human head and neck cancer cells through the inhibition of Integrin β1/FAK/Akt signaling

MiR-34a was identified as one of the down-regulated micro-RNAs (miRs) in human colorectal cancer 5-fluorouracil (5-FU)-resistant DLD-1 cells compared with those in the parental DLD-1 cells. Exposure to 5-FU at 30 μM activated phosphoinositide 3-kinase (PI3K)/Akt signaling markedly from 12h up to 48 h in the 5-FU-resistant cells compared with that in the parental cells and resulted in an overt difference in growth at those times. Furthermore, the expression of miR-34a in the 5-FU-resistant cells was sustained at a low-level, whereas it was up-regulated in the parental cells after the 5-FU treatment. Sirt1, which is one of the target genes for miR-34a and related to drug-resistance, was strikingly up-regulated in the 5-FU-resistant cells. The ectopic expression of miR-34a in the 5-FU-resistant cells inhibited growth, as in the parental cells, and attenuated the resistance to 5-FU through the down-regulation of Sirt1 and E2F3. Moreover, the silencing of Sirt1 significantly canceled the resistance to 5-FU in the 5-FU-resistant cells. These findings suggest that miR-34a targeting the Sirt1 and E2F3 genes could negatively regulate, at least in part, the resistance to 5-FU in human colorectal cancer DLD-1 cells.     

Knockdown of CEACAM19 suppresses human gastric cancer through inhibition of PI3K/Akt and NF-κB

Gastric cancer directly affects the quality of human life worldwide. Some members, which belong to carcinoembryonic antigen-related cell adhesion molecule (CEACAM) subfamily, are deregulated in tumors. Of the subfamily, CEACAM19, a new member was the research object. Our study sought to explore the potential role of CEACAM19 in gastric cancer. According to the immunohistochemistry (IHC), RT-PCR and Western blot, CEACAM19 was over-expressed in gastric cancer tissues and cells. Moreover, the Western blot analysis showed that the expression of MMP2 and MMP9 was inhibited in CEACAM19 knockdown gastric cancer cells. Meanwhile, in SGC-7901 and MGC-803?cells, the knockdown of CEACAM19 reduced proliferation, migration and invasion. Additionally, the Western blot assay revealed that the phosphorylation levels of Akt and p65 were declined by the knockdown of CEACAM19. Furthermore, the influence of CEACAM19 knockdown was confirmed by the studies in vivo. Collectively, our results revealed that the CEACAM19 knockdown prevented the gastric cancer progression likely related to inactivating the PI3K/Akt and NF-κB signaling pathways. Our findings provided insights into a promising biomarker of gastric cancer and the potential molecule clues for the prevention of gastric cancer.     

Defective NF-κB signaling in metastatic head and neck cancer cells leads to enhanced apoptosis by double-stranded RNA

Ligands to several Toll-like receptors (TLR), which mediate innate immune responses and chronic inflammation have been used as adjuvants to immunotherapy to enhance their antitumor activity. In particular, double-stranded RNAs that are cognate ligands of TLR3 have been used to trigger proapoptotic activity in cancer cells. However, a mechanistic understanding of TLR3-mediated apoptosis and its potential involvement in controlling tumor metastasis has been lacking. In this study, we used paired cell lines and fresh tumor specimens, derived from autologous primary and metastatic head and neck squamous cell carcinoma, to investigate the role of TLR3 signaling in metastatic progression. Compared with primary tumor cells, metastatic tumor cells were highly sensitive to TLR3-mediated apoptosis after double-stranded RNA treatment. Enhanced apoptosis in metastatic cells was dependent on double-stranded RNA and TLR3 and also the TLR3 effector signaling protein TRIF. Downstream responses requiring NF-κB were critical for apoptosis in metastatic cells, the defects in which could be resuscitated by alternative pathways of NF-κB activation. By elucidating how TLR3 ligands trigger apoptosis in metastatic cells, our findings suggest insights into how to improve their clinical use.     

Berberine suppresses in vitro migration and invasion of human SCC-4 tongue squamous cancer cells through the inhibitions of FAK,IKK, NF-κB,u-PA and MMP-2 and -9

There is increasing evidence that urokinase-type plasminogen activator (u-PA) and matrix metalloproteinases (MMPs) play an important role in cancer metastasis and angiogenesis. Inhibition of u-PA and MMPs could suppress migration and invasion of cancer cells. Berberine, one of the main constituents of the plant Rhizoma coptidis, is a type of isoquinoline alkaloid, reported to have anti-cancer effects in different human cancer cell lines. There is however, no available information on effects of berberine on migration and invasion of human tongue cancer cells. Here, we report that berberine inhibited migration and invasion of human SCC-4 tongue squamous carcinoma cells. This action was mediated by the p-JNK, p-ERK, p-p38, IκK and NF-κB signaling pathways resulting in inhibition of MMP-2 and -9 in human SCC-4 tongue squamous carcinoma cells. Our Western blowing analysis also showed that berberine inhibited the levels of urokinase-plasminogen activator (u-PA). These results suggest that berberine down-regulates u-PA, MMP-2 and -9 expressions in SCC-4 cells through the FAK, IKK and NF-κB mediated pathways and a novel function of berberine is to inhibit the invasive capacity of malignant cells.     

LGR5 promotes the proliferation and tumor formation of cervical cancer cells through the Wnt/β-catenin signaling pathway

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a seven transmembrane receptor known as a potential stem cell marker for intestinal crypts and hair follicles, has recently been found to be overexpressed in some types of human cancers. However, the role of LGR5 in cervical cancer remains unclear. In this study, the expression of LGR5 gradually increases from normal cervix to cervical cancer in situ and to cervical cancers as revealed by immunohistochemistry and western blot analyses. Through knocking down or overexpressing LGR5 in SiHa and HeLa cells, the expression level of LGR5 was found to be positively related to cell proliferation in vitro and to tumor formation in vivo. Further investigation indicated that LGR5 protein could significantly promote the acceleration of cell cycle. Moreover, the TOP-Flash reporter assay and western blot for β-catenin, cyclinD1, and c-myc proteins, target genes of the Wnt/β-catenin pathway, indicated that LGR5 significantly activated Wnt/β-catenin signaling. Additionally, the blockage of Wnt/β-catenin pathway resulted in a significant inhibition of cell proliferation induced by LGR5. Taken together, these results demonstrate that LGR5 can promote proliferation and tumor formation in cervical cancer cells by activating the Wnt/β-catenin pathway.     

Perioperative biobehavioral interventions to prevent cancer recurrence through combined inhibition of β-adrenergic and cyclooxygenase 2 signaling

Evidence suggests that excess perioperative activation of the sympathetic nervous system and the consequent release of catecholamines (ie, epinephrine and norepinephrine) in the context of cancer surgery and inflammation may significantly facilitate prometastatic processes. This review first presents biomedical processes that make the perioperative timeframe pivotal in determining long-term cancer outcomes nonproportionally to its short duration (days to weeks). Then, it analyzes the various mechanisms via which the excess release of catecholamines can facilitate the progression of cancer metastases in this context by directly affecting the malignant tissues and by regulating, via indirect pathways, immunological and other mechanisms that affect metastatic progression in the tumor microenvironment and systemically. In addition, this review addresses the need to supplement β-adrenoreceptor blockade with cyclooxygenase 2 inhibition, especially during surgery and shortly thereafter, because similar mechanisms are simultaneously activated by surgery-induced inflammatory responses. Importantly, this review presents translational and clinical evidence showing that perioperative β-adrenoreceptor blockade and cyclooxygenase 2 inhibition can reduce the prometastatic process and cancer recurrence, and the clinical feasibility and safety of this approach are demonstrated as well. Lastly, alternative psychophysiological approaches to the use of β-adrenergic blockers are presented because a substantial portion of patients have medical contraindications to this pharmacological treatment. The adaptation of existing psychophysiological interventions to the perioperative period and principles for constructing new approaches are discussed and exemplified. Overall, pharmacobehavioral interventions, separately or in combination, could transform the perioperative timeframe from being a prominent facilitator of metastatic progression to an opportunity for arresting or eliminating residual disease, potentially improving long-term survival rates in cancer patients.     

Acquisition of EMT phenotype in the gefitinib-resistant cells of a head and neck squamous cell carcinoma cell line through Akt/GSK-3β/snail signalling pathway

Background:

Epithelial mesenchymal transition (EMT) is known to be associated with chemoresistance as well as increased invasion/metastasis. However, the relationship between EMT and resistance to an epidermal growth factor receptor (EGFR) -targeting drug in head and neck squamous cell carcinoma (HNSCC) remains unknown. In this study, we investigated the acquisition of EMT by gefitinib in HNSCC cell line (UMSCC81B).

Methods:

We isolated fibroblastoid variant (81B-Fb) from gefitinib-resistant UMSCC81B-GR3 cells obtained after increasing the doses of gefitinib treatment in vitro and examined EMT and its underlying mechanism.

Result:

81B-Fb cells exhibited fibroblast-like morphology, increased motility, loss of E-cadherin, acquisition of vimentin and snail expression. In 81B-Fb cells, downregulation of EGFR, which is mediated by increased ubiquitination, and activation of downstream protein kinase B (Akt), glycogen synthase kinase-beta (GSK-3β) signalling and upregulation of snail expression were observed compared with UMSCC81B cells. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, but not U0126, suppressed foetal bovine serum or heregulin-β1-induced phosphorylation of Akt/GSK-3β and snail expression together with the inhibition of 81B-Fb cell motility. Furthermore, forced expression of EGFR resulted in partial restoration of gefitinib sensitivity and reversal of EMT.

Conclusion:

These results suggest that EMT in the gefitinib-resistant cells is mediated by the downregulation of EGFR and compensatory activation of Akt/GSK-3β/snail pathway.     

First-line pembrolizumab ± chemotherapy for recurrent/metastatic head and neck cancer: Japanese subgroup of KEYNOTE-048

Background

Here, we report the results of the Japanese subgroup of the phase 3 KEYNOTE-048 study of pembrolizumab alone, pembrolizumab plus platinum and 5-fluorouracil (pembrolizumab–chemotherapy), or cetuximab plus platinum and 5-fluorouracil (EXTREME) in previously untreated recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC).

Methods

Primary end points were overall survival (OS) and progression-free survival (PFS). Efficacy was evaluated in patients with PD-L1 combined positive score (CPS) ≥ 20 and ≥ 1 and the total Japanese subgroup (n = 67).

Results

At data cutoff (25 February 2019), pembrolizumab led to longer OS versus EXTREME in the PD-L1 CPS ≥ 20 subgroup (median, 28.2 vs. 13.3 months; HR, 0.29 [95% CI 0.09–0.89]) and to similar OS in the total Japanese (23.4 vs. 13.6 months; HR, 0.51 [95% CI 0.25–1.05]) and CPS ≥ 1 subgroups (22.6 vs. 15.8 months; HR, 0.66 [95% CI 0.31–1.41]). Pembrolizumab–chemotherapy led to similar OS versus EXTREME in the PD-L1 CPS ≥ 20 (median, 18.1 vs. 15.8 months; HR, 0.72 [95% CI 0.23–2.19]), CPS ≥ 1 (12.6 vs. 15.8 months; HR, 1.19 [95% CI 0.55–2.58]), and total Japanese subgroups (12.6 vs. 13.3 months; unadjusted HR, 1.10 [95% CI 0.55–2.22]). Median PFS was similar for pembrolizumab and pembrolizumab–chemotherapy versus EXTREME in all subgroups. Grades 3–5 treatment-related adverse events occurred in 5 (22%), 19 (76%), and 17 (89%) patients receiving pembrolizumab, pembrolizumab–chemotherapy, and EXTREME, respectively. One patient receiving pembrolizumab–chemotherapy died because of treatment-related pneumonitis.

Conclusion

These results support the use of first-line pembrolizumab and pembrolizumab–chemotherapy for Japanese patients with R/M HNSCC.

Clinical trial registry ClinicalTrials.gov, NCT02358031.

     

Lunasin potentiates the effect of oxaliplatin preventing outgrowth of colon cancer metastasis, binds to α5β1 integrin and suppresses FAK/ERK/NF-κB signaling

The effect of lunasin on colon cancer metastasis was studied using three human colon cancer cell lines in vitro and a liver metastasis model of colon cancer in vivo. Lunasin bound with α₅β₁ integrin and internalized into the nucleus of KM12L4 human colon cancer cells. Lunasin (10 μM) inhibited the activation of focal adhesion kinase (FAK) by 28%, 39% and 60% in RKO, HCT-116 and KM12L4 human colon cancer cells, respectively. Lunasin caused an increase in the expression of the inhibitor of kappa B alpha (IκB-α), a decrease in nuclear p50 NF-κB and a reduction in the migration of cancer cells. Lunasin (4 mg/kg bw) inhibited metastasis and potentiated the effect of oxaliplatin by reducing the expression of proliferating cell nuclear antigen. Liver metastatic nodules were reduced from 28 (PBS) to 14 (lunasin, P = 0.047) while combination of lunasin and oxaliplatin to 5 (P = 0.004). The tumor burden was reduced from 0.13 (PBS) to 0.10 (lunasin, P = 0.039) to 0.04 (lunasin + oxaliplatin, P < 0.0001). Moreover, lunasin potentiated the effect of oxaliplatin in modifying expression of proteins involved in apoptosis and metastasis including Bax, Bcl-2, IKK-α and p-p65. Lunasin inhibited metastasis of human colon cancer cells by direct binding with α₅β₁ integrin suppressing FAK/ERK/NF-κB signaling, and potentiated the effect of oxaliplatin in preventing the outgrowth of metastasis.     

NNK promotes migration and invasion of lung cancer cells through activation of c-Src/PKCι/FAK loop

Cigarette smoking, either active or passive, is the most important risk factor in the development of human lung cancer. Mounting evidence indicates that cigarette smoke constituents not only contribute to tumorigenesis but also may increase the spread of cancer in the body. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen. NNK, an important component in cigarette smoke, may also promote tumor metastasis by regulating cell motility. Here we found that NNK can induce activation of a functionally interdependent protein kinase cascade, including c-Src, PKCι and FAK, in association with increased migration and invasion of human lung cancer cells. c-Src, PKCι and FAK are extensively co-localized in the cytoplasm. Treatment of cells with α₇ nAChR specific inhibitor α-bungarotoxin (α-BTX) blocks NNK-stimulated activation of c-Src, PKCι and FAK and suppresses cell migration and invasion. Intriguingly, NNK enhances c-Src/PKCι and PKCι/FAK bindings, indicating a potential mechanism by which these kinases activate each other. Specific disruption of c-Src, PKCι or FAK expression by RNA interference significantly reduces NNK-induced cell migration and invasion. These findings suggest that NNK-induced migration and invasion may occur in a mechanism through activation of a c-Src/PKCι/FAK loop, which can contribute to metastasis and/or development of human lung cancer.     

Elevated TAK1 augments tumor growth and metastatic capacities of ovarian cancer cells through activation of NF-κB signaling

Transforming growth factor (TGF)-β-activating kinase 1 (TAK1) is a serine/threonine kinase which is frequently associated with human cancer progression. However, its functional role in tumorigenesis is still controversial. Here, we report that TAK1 enhances the oncogenic capacity of ovarian cancer cells through the activation of NF-κB signaling. We found that TAK1 is frequently upregulated and significantly associated with high-grade and metastatic ovarian cancers. Mechanistic studies showed that Ser412 phosphorylation is required for TAK1 in activating NF-κB signaling and promotes aggressiveness of ovarian cancer cells. Conversely, suppression of TAK1 activity by point mutation at Ser412, RNAi mediated gene knockdown or TAK1 specific inhibitor ((5Z) -7-Oxozeaenol) remarkably impairs tumor growth and metastasis in ovarian cancer in vitro and in vivo. Our study underscores the importance of targeting TAK1 as a promising therapeutic approach to counteract the ovarian cancer progression.     

A vitamin D receptor-alkylating derivative of 1α,25-dihydroxyvitamin D3 inhibits growth of human kidney cancer cells and suppresses tumor growth

1,25-Dihydroxyvitamin D? [1,25(OH)?D?] has shown strong promise as an antiproliferative agent in several malignancies, yet its therapeutic use has been limited by its toxicity leading to search for analogues with antitumor property and low toxicity. In this study, we evaluated the in vitro and in vivo properties of 1,25-dihydroxyvitamin D?-3-bromoacetate [1,25(OH)?D?-3-BE], an alkylating derivative of 1,25(OH)?D?, as a potential therapeutic agent for renal cancer. Dose response of 1,25(OH)?D?-3-BE in 2 kidney cancer cell lines was evaluated for its antiproliferative and apoptotic properties, and mechanisms were evaluated by Western blot and FACS analyses. Therapeutic potential of 1,25(OH)?D?-3-BE was assessed both by determining its stability in human serum and by evaluating its efficacy in a mouse xenograft model of human renal tumor. We observed that 1,25(OH)?D?-3-BE is significantly more potent than an equivalent concentration of 1,25(OH)?D? in inhibiting growth of A498 and Caki 1 human kidney cancer cells. 1,25(OH)?D?-3-BE-mediated growth inhibition was promoted through inhibition of cell-cycle progression by downregulating cyclin A and induction of apoptosis by stimulating caspase activity. Moreover, 1,25(OH)?D?-3-BE strongly inhibited Akt phosphorylation and phosphorylation of its downstream target, caspase-9. 1,25(OH)?D?-3-BE seemed to be stable in human serum. In xenograft mouse model of human renal tumor, 1,25(OH)?D?-3-BE was more potent at reducing tumor size than 1,25(OH)?D?, which was accompanied by an increase in apopotosis and reduction of cyclin A staining in the tumors. These results suggest a translational potential of this compound as a therapeutic agent in renal cell carcinoma. Data from this study and extensive studies of vitamin D for the prevention of many malignancies support the potential of 1,25(OH)?D?-3-BE for preventing renal cancer and the development of relevant in vivo prevention models for assessing this potential, which do not exist at present.     

Synthetic triterpenoids inhibit growth and induce apoptosis in human glioblastoma and neuroblastoma cells through inhibition of prosurvival Akt,NF-<Emphasis Type="Italic">κ</Emphasis>B and Notch1 signaling

Glioblastomas are high-risk primary brain tumors that are generally unresponsive or only weakly responsive to the currently available antineoplastic agents. Thus novel therapeutic strategies and agents are urgently needed to treat these incurable cancers. Oleanolic acid and ursolic acid are naturally occurring triterpenoids that have been used in traditional Asian medicine as anti-inflammatory and anti-cancer agents. Recently, synthetic oleanolic acid triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its C-28 methyl ester (CDDO-Me) and C-28 imidazole (CDDO-Im) derivatives have been shown to exhibit potent antitumor activity against diverse types of tumor cell lines, including leukemia, multiple myeloma, osteosarcoma, breast, lung, and pancreatic cancer cell lines; however, the anticancer activity of these agents for brain tumors has not been reported. In the present study, we investigated the apoptosis-inducing activity of CDDOs in glioblastoma (U87MG, U251MG) and neuroblastoma (SK-N-MC) cell lines. Cell growth/viability (MTS) and cytotoxicity (LDH release) assays demonstrated that glioblastoma cell lines are least sensitive to CDDO, but are highly sensitive to CDDO-Me and CDDO-Im at concentrations of 2.5–10 μM. CDDO-Im and CDDO-Me were equipotenent in their growth inhibitory activity. The primary mode of tumor cell destruction was apoptosis as demonstrated by significant increase in the number of hypo-diploid (sub-G0) cells and annexin V-FITC binding. Induction of apoptosis was associated with the activation of procaspases-3, -8, and -9, mitochondrial depolarization and the release of cytochrome c from mitochondria. Furthermore, CDDO-Me inhibited the levels of anti-apoptotic and prosurvival p-Akt, NF-κB (p65) and Notch1 signaling molecules. These studies provide rationale for clinical evaluation of these novel agents for the management of lethal brain neoplasms.     

Glycogen synthase kinase-3β inhibition depletes the population of prostate cancer stem/progenitor-like cells and attenuates metastatic growth

Cancer cells with stem or progenitor properties play a pivotal role in the initiation, recurrence and metastatic potential of solid tumors, including those of the human prostate. Cancer stem cells are generally more resistant to conventional therapies thus requiring the characterization of key pathways involved in the formation and/or maintenance of this malignant cellular subpopulation.To this end, we identified Glycogen Synthase Kinase-3β (GSK-3β) as a crucial kinase for the maintenance of prostate cancer stem/progenitor-like cells and pharmacologic inhibition of GSK-3β dramatically decreased the size of this cellular subpopulation. This was paralleled by impaired clonogenicity, decreased migratory potential and dramatic morphological changes. In line with our in vitro observations, treatment with a GSK-3β inhibitor leads to a complete loss of tumorigenicity and a decrease in metastatic potential in preclinical in vivo models. These observed anti-tumor effects appear to be largely Wnt-independent as simultaneous Wnt inhibition does not reverse the observed antitumor effects of GSK-3β blockage. We found that GSK-3β activity is linked to cytoskeletal protein F-actin and inhibition of GSK-3β leads to disturbance of F-actin polymerization. This may underlie the dramatic effects of GSK-3β inhibition on prostate cancer migration. Furthermore, GSK-3β inhibition led to strongly decreased expression of several integrin types including the cancer stem cell-associated α2β1 integrin. Taken together, our mechanistic observations highlight the importance of GSK-3β activity in prostate cancer stemness and may facilitate the development of novel therapy for advanced prostate cancer.     

All-trans-retinoic acid inhibits growth of head and neck cancer stem cells by suppression of Wnt/β-catenin pathway

Differentiation therapy is a novel approach to eradicate cancer stem cells (CSCs), including head and neck squamous carcinoma CSC (HNSC CSC). All-trans-retinoic acid (ATRA) is a potent differentiating agent. We studied the anti-tumour effect of ATRA on HNSC CSC. HNSC CSCs were differentiated by ATRA in a serum-free conditioned medium. The effect of differentiation on tumour growth was assessed in vitro and in vivo, and chemosensitisation was examined using a colorimetric viability assay. In addition, the involvement of Wnt/β-catenin signalling as an underlying mechanism of the anti-tumour effect of retinoic acid (RA) on HNSC CSCs was assessed. ATRA suppressed the expression of the stem cell markers Oct4, Sox2, Nestin and CD44 in HNSC CSCs and inhibited the proliferation of HNSC CSCs in vitro and in vivo. Furthermore, ATRA treatment augmented the chemosensitising effects of cisplatin. The anti-tumour effects of ATRA may be associated with down-regulation of Wnt/β-catenin signalling. In conclusion, ATRA may be potentially valuable in treatment of HNSC CSC, especially in combination with cisplatin.     

PRL-3 promotes the motility, invasion, and metastasis of LoVo colon cancer cells through PRL-3-integrin β1-ERK1/2 and-MMP2 signaling

Background  

Phosphatase of regenerating liver-3 (PRL-3) plays a causative role in tumor metastasis, but the underlying mechanisms are not well understood. In our previous study, we observed that PRL-3 could decrease tyrosine phosphorylation of integrin β1 and enhance activation of ERK1/2 in HEK293 cells. Herein we aim to explore the association of PRL-3 with integrin β1 signaling and its functional implications in motility, invasion, and metastasis of colon cancer cell LoVo.