Calpain activation through galectin-3 inhibition sensitizes prostate cancer cells to cisplatin treatment

Prostate cancer will develop chemoresistance following a period of chemotherapy. This is due, in part, to the acquisition of antiapoptotic properties by the cancer cells and, therefore, development of novel strategies for treatment is of critical need. Here, we attempt to clarify the role of the antiapoptotic molecule galectin-3 in prostate cancer cells using siRNA and antagonist approaches. The data showed that Gal-3 inhibition by siRNA or its antagonist GCS-100/modified citrus pectin (MCP) increased cisplatin-induced apoptosis of PC3 cells. Recent studies have indicated that cisplatin-induced apoptosis may be mediated by calpain, a calcium-dependent protease, as its activation leads to cleavage of androgen receptor into an androgen-independent isoform in prostate cancer cells. Thus, we examined whether calpain activation is associated with the Gal-3 function of regulating apoptosis. Here, we report that Gal-3 inhibition by siRNA or GCS-100/MCP enhances calpain activation, whereas Gal-3 overexpression inhibits it. Inhibition of calpain using its inhibitor and/or siRNA attenuated the proapoptotic effect of Gal-3 inhibition, suggesting that calpain activation may be a novel mechanism for the proapoptotic effect of Gal-3 inhibition. Thus, a paradigm shift for treating prostate cancer is suggested whereby a combination of a non-toxic anti-Gal-3 drug together with a toxic chemotherapeutic agent could serve as a novel therapeutic modality for chemoresistant prostate cancers.     

Pooling analysis reveals that galectin-1 is a reliable prognostic biomarker in various cancers

Galectin-1 is reported to be upregulated in various human cancers. However, the relationship between galectin-1 expression and cancer prognosis has not been systematically assessed. In this study, we searched PubMed, Web of Science, and Embase to collect all relevant studies and a meta-analysis was performed. We found that increased galectin-1 expression was associated with tumor size (odds ratio [OR] = 1.75; 95% confidence interval [CI]: 1.06–2.89; p = 0.029), clinical stage (OR = 3.89; 95% CI: 2.40–6.31; p < 0.001), and poorer differentiation (OR = 1.39; 95% CI: 1.14–1.69; p = 0.001), but not with age (OR = 1.07; 95% CI: 0.82–1.39; p = 0.597), sex (OR = 0.89; 95% CI: 0.74–1.07; p = 0.202), or lymph node metastasis (OR = 2.57; 95% CI: 0.98–6.78; p = 0.056). In addition, we found that high galectin-1 expression levels were associated with poor overall survival (HR = 2.12; 95% CI: 1.71–2.64; p < 0.001). The results were further validated using The Cancer Genome Atlas data set. Moreover, high galectin-1 expression was significantly associated with disease-free survival (hazard ratio [HR] = 1.60; 95% CI: 1.17–2.19; p = 0.003), progression-free survival (HR = 1.93; 95% CI: 1.65–2.25; p < 0.001), and cancer-specific survival (HR = 1.82; 95% CI: 1.30–2.55; p < 0.001). Our meta-analysis demonstrated that galectin-1 might be a useful common biomarker for predicting prognosis in patients with cancer.     

Dedifferentiated Schwann cells promote perineural invasion mediated by the PACAP paracrine signalling in cervical cancer

Perineural invasion (PNI) has emerged as a key pathological feature and be considered as a poor prognostic factor in cervical cancer. However, the underlying molecular mechanisms are largely unknown. Here, PNI status of 269 cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) samples were quantified by using whole-slide diagnostic images obtained from The Cancer Genome Atlas. Integrated analyses revealed that PNI was an indicative marker of poorer disease-free survival for CESC patients. Among the differentially expressed genes, ADCYAP1 were identified. Clinical specimens supported that high expression of PACAP (encoded by ADCYAP1) contributed to PNI in CESC. Mechanistically, PACAP, secreted from cervical cancer cells, reversed myelin differentiation of Schwann cells (SCs). Then, dedifferentiated SCs promoted PNI by producing chemokine FGF17 and by degrading extracellular matrix through secretion of Cathepsin S and MMP-12. In conclusion, this study identified PACAP was associated with PNI in cervical cancer and suggested that tumour-derived PACAP reversed myelin differentiation of SCs to aid PNI.     

Resveratrol significantly inhibits the occurrence and development of cervical cancer by regulating phospholipid scramblase 1

Cervical cancer (CC) is one of the most common female malignancies, and resveratrol is a polyphenol isolated from the skins of grapes, which has been reported to significantly alter the cellular physiology of tumor cells. However, little is known about the role of phospholipid scramblase 1 (PLSCR1) in pathogenesis of CC. Here, we demonstrated that resveratrol could significantly inhibit both the growth of HeLa cells and expression of PLSCR1. These results suggest that resveratrol-mediated cell growth inhibition can be regulated by PLSCR1.     

Effects of galectin-1 on regulation of progesterone production in granulosa cells from pig ovaries in vitro

The detection of galectin-1 (gal-1) in pig granulosa cell lysates by immunoblotting and its cytosolic as well as membrane-associated localization prompted us to study its effects on cell proliferation and regulation of progesterone synthesis. The lectin stimulated the proliferation of granulosa cells from pig ovaries cultured in serum-free medium. Gal-1 inhibited the FSH-stimulated progesterone synthesis of granulosa cells. This inhibitory effect was strongly reduced by the disaccharidic competitor lactose at 30 mM. The absence of inhibitory effects on dibutyryl-cAMP (db-cAMP), forskolin, and pregnenolone-enhanced cellular progesterone synthesis suggests that gal-1interferes with the receptor-dependent mechanism of FSH-stimulated progesterone production. In FSH-stimulated granulosa cells, western blot analysis revealed the gal-1-mediated suppression of the cytochrome P450-dependent cholesterol side chain cleavage enzyme (P450(SCC)) that catalyzes the conversion of cholesterol to pregnenolone. In the presence of 30 mM lactose, the gal-1-reduced P450(SCC) expression was prevented. Strongly reduced mRNA levels were recorded for P450(SCC) and 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) when FSH-stimulated granulosa cells were cultured in the presence of gal-1. We conclude that gal-1 exerts its inhibitory effect on steroidogenic activity of granulosa cells by interfering the hormone-receptor interaction resulting in decreased responses to FSH stimulation.     

Apoptotic effect of methanol extract of Picrasma quassioides by regulating specificity protein 1 in human cervical cancer cells

In the present study, we examined the effects of methanol extracts of Picrasma quassioides (MEPQ) on apoptosis in human cervical cancer cells. The results showed that MEPQ decreased the viability and induced caspase‐dependent apoptosis in HEp‐2 cells. MEPQ decreased specificity protein 1 (Sp1) in HEp‐2 cells, whereas Sp1 mRNA was not changed. We found that MEPQ reduced Sp1 protein through proteasome‐dependent protein degradation, but not the inhibition of protein synthesis. Also, MEPQ increased the expressions of Bad and truncated Bid (t‐Bid) but did not alter other Bcl‐2 family members. The knock‐down of Sp1 by both Sp1 interfering RNA and Mithramycin A, Sp1 specific inhibitor clearly increased Bad and t‐Bid expression to decrease cell viability and induce apoptosis. In addition, MEPQ inhibited cell viability and induced apoptotic cell death through the modulation of Sp1 in KB cells. These results suggest that MEPQ may be a potential anticancer agent for human cervical cancer. Copyright © 2013 John Wiley & Sons, Ltd.     

Cell-surface galectin-3 confers resistance to TRAIL by impeding trafficking of death receptors in metastatic colon adenocarcinoma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis and preferentially kills tumor cells by engaging specific glycosylated death receptors, resulting in the internalization of ligand/receptor complexes and recruitment of the initiator caspase-8 to an activation platform known as the death-inducing signaling complex (DISC). However, emergence of TRAIL-resistant sub-populations may contribute to therapeutic failure. To investigate resistance mechanisms, we isolated a stable TRAIL-resistant sub-population of the metastatic colon cancer cell line LS-LIM6, designated LIM6-TR. LIM6-TR cells are impaired in endocytosis of TRAIL/death receptors complexes and failed to recruit/activate caspase-8 to the DISC upon TRAIL stimulation. Differential activation of Wnt and JNK pathways is not responsible for acquisition of TRAIL resistance. LIM6-TR cells display a marked increase in cell-surface expression of galectin-3, an endogenous lectin, which co-localizes with and binds death receptors. Silencing of galectin-3 restores TRAIL sensitivity and promotes TRAIL-mediated endocytosis of TRAIL/death receptors complexes. Inhibitors of galectin-3 and glycosylation also re-sensitize LIM6-TR to TRAIL and restore internalization of ligand/receptors complexes. These studies identify a novel TRAIL-resistance mechanism in which galectin-3 impedes trafficking of death receptor by anchoring them in glycan nano-clusters, blocking the execution of the apoptosis signal.     

Overexpression of MALAT1 contributes to cervical cancer progression by acting as a sponge of miR-429

Cervical cancer remains a malignant type of tumor and is the fourth leading cause of cancer-related death among females. MALAT1 has been identified as a tumor oncogene in various cancers. Our present study aimed to explore the biological role of MALAT1 in cervical cancer. We observed that MALAT1 was significantly upregulated in human cervical cancer cell lines compared with the ectocervical epithelial cells. MALAT1 was repressed by transfection with LV-shMALAT1, whereas increased by LV-MALAT1 in HeLa and Caski cells. Silencing of MALAT1 obviously reduced cervical cell viability, induced cell apoptosis, and repressed cell invasion capacity. Conversely, overexpression of MALAT1 exhibited an opposite phenomenon. Furthermore, miR-429 was predicted as a direct target of MALAT1, and it was dramatically decreased in cervical cancer cells. It has been shown that miR-429 plays a crucial role in cervical cancer progression. In our current study, the targeting correlation between MALAT1 and miR-429 was confirmed by luciferase reporter assays and RIP experiments. Finally, in vivo animal models were established, and we indicated that MALAT1 inhibited cervical cancer progression via targeting miR-429. These findings revealed that MALAT1 can sponge miR-429 and regulate cervical cancer pathogenesis in vivo and in vitro. In conclusion, we indicated that the MALAT1/miR-429 axis was involved in cervical cancer development.     

Synthesis of an endogeneous lectin, galectin-1, by human endothelial cells is up-regulated by endothelial cell activation

The pattern of expression of an endogenous lectin, galectin-1, was examined in human lymphoid tissue. Galectin-1 was detected in the endothelial cells lining specialized vessels, termed high endothelial venules, in activated lymphoid tissue, but not in a resting lymph node. Cultured endothelial cells (human aortic and umbilical vein endothelial cells (HAECs and HUVECs)) expressed galectin-1. Activation of the cultured endothelial cells increased the level of galectin-1 expression, as determined by ELISA, Northern blot analysis and high throughput cDNA sequencing. These results suggest that galectin-1 expressed by endothelial cells may bind to and affect the trafficking of cells emigrating from blood into tissues.     

CLEFMA induces intrinsic and extrinsic apoptotic pathways through ERK1/2 and p38 signalling in uterine cervical cancer cells

Although concurrent chemoradiotherapy is the cornerstone of treatment for locally advanced or recurrent uterine cervical cancer, treatment fails at a high rate. Therefore, the development of novel targeting agents is critical. This study investigated the action of CLEFMA, a potent, synthetic curcumin derivative, on cervical cancer cells and its mechanism of action. We found that CLEFMA negatively regulated the viability of cervical cancer cells, involving induction of cell apoptosis. Cleaved caspase-3, cleaved poly(adenosine diphosphate-ribose) polymerase, cleaved caspase-8, and cleaved caspase-9 expression were increased by treatment with CLEFMA. After U0126 (ERK1/2 inhibitor) and SB203580 (p38 inhibitor) were applied as cotreatment with CLEFMA, the expression of cleaved caspase-8, -9, and -3 was reduced significantly. In conclusion, CLEFMA activates both extrinsic and intrinsic apoptotic pathways through ERK1/2 and p38 signal transduction in cervical cancer cells.     

Mechanisms of radioresistance and the underlying signaling pathways in colorectal cancer cells

Radiotherapy is one of the most common modalities for the treatment of a wide range of tumors, including colorectal cancer (CRC); however, radioresistance of cancer cells remains a major limitation for this treatment. Following radiotherapy, the activities of various cellular mechanisms and cell signaling pathways are altered, resulting in the development of radioresistance, which leads to therapeutic failure and poor prognosis in patients with cancer. Furthermore, even though several inhibitors have been developed to target tumor resistance, these molecules can induce side effects in nontumor cells due to low specificity and efficiency. However, the role of these mechanisms in CRC has not been extensively studied. This review discusses recent studies regarding the relationship between radioresistance and the alterations in a series of cellular mechanisms and cell signaling pathways that lead to therapeutic failure and tumor recurrence. Our review also presents recent advances in the in vitro/in vivo study models aimed at investigating the radioresistance mechanism in CRC. Furthermore, it provides a relevant biochemical basis in theory, which can be useful to improve radiotherapy sensitivity and prolong patient survival.     

LncRNA TP73-AS1 is a novel regulator in cervical cancer via miR-329-3p/ARF1 axis

Cervical cancer holds one of the highest morbidity and mortality in various types of cancers. It even leads to the most number of cancer-related deaths of women. A lot of research has indicated that the anomalous expression of long noncoding RNAs (lncRNAs) would induce carcinogenesis and is associated with poor prognosis of patients with cancer. However, the function and mechanism of many lncRNAs still call for further research. Tumor Protein P73 Antisense RNA 1 (TP73-AS1) is no exception. LncRNA TP73-AS1 has been found to promote cancer progressions in various cancers. It is upregulated in cervical cancer cells. The proliferation and migration ability of cervical cancer cells can also be boosted by TP73-AS1 in return. Meanwhile, miRNA-329-3p is downregulated in cervical cancer cells and could bind with both TP73-AS1 and ADP Ribosylation Factor 1 (ARF1). TP73-AS1 inhibited miR-329-3p expression while miR-329-3p inhibited ARF1 expression. More importantly, TP73-AS1 can positively regulate ARF1 expression. Based on all these experiments, TP73-AS1 regulates ARF1 expression by competitively binding with miR-329-3p, thus regulating cervical cancer progression. Further rescue assays confirmed TP73-AS1 regulates cervical cell proliferation and migration via miR-329-3p/ARF1. TP73-AS1 might serve as a novel regulator in cervical cancer.     

Cyclin D1 overexpression perturbs DNA replication and induces replication-associated DNA double-strand breaks in acquired radioresistant cells

Fractionated radiotherapy (RT) is widely used in cancer treatment, because it preserves normal tissues. However, repopulation of radioresistant tumors during fractionated RT limits the efficacy of RT. We recently demonstrated that a moderate level of long-term fractionated radiation confers acquired radioresistance to tumor cells, which is caused by DNA-PK/AKT/GSK3β-mediated cyclin D1 overexpression. The resulting cyclin D1 overexpression leads to forced progression of the cell cycle to S-phase, concomitant with induction of DNA double-strand breaks (DSBs). In this study, we investigated the molecular mechanisms underlying cyclin D1 overexpression-induced DSBs during DNA replication in acquired radioresistant cells. DNA fiber data demonstrated that replication forks progressed slowly in acquired radioresistant cells compared with corresponding parental cells in HepG2 and HeLa cell lines. Slowly progressing replication forks were also observed in HepG2 and HeLa cells that overexpressed a nondegradable cyclin D1 mutant. We also found that knockdown of Mus81endonuclease, which is responsible for resolving aberrant replication forks, suppressed DSB formation in acquired radioresistant cells. Consequently, Mus81 created DSBs to remove aberrant replication forks in response to replication perturbation triggered by cyclin D1 overexpression. After treating cells with a specific inhibitor for DNA-PK or ATM, apoptosis rates increased in acquired radioresistant cells but not in parental cells by inhibiting the DNA damage response to cyclin D1-mediated DSBs. This suggested that these inhibitors might eradicate acquired radioresistant cells and improve fractionated RT outcomes.     

The glycan-binding protein galectin-1 controls survival of epithelial cells along the crypt-villus axis of small intestine

Intestinal epithelial cells serve as mechanical barriers and active components of the mucosal immune system. These cells migrate from the crypt to the tip of the villus, where different stimuli can differentially affect their survival. Here we investigated, using in vitro and in vivo strategies, the role of galectin-1 (Gal-1), an evolutionarily conserved glycan-binding protein, in modulating the survival of human and mouse enterocytes. Both Gal-1 and its specific glyco-receptors were broadly expressed in small bowel enterocytes. Exogenous Gal-1 reduced the viability of enterocytes through apoptotic mechanisms involving activation of both caspase and mitochondrial pathways. Consistent with these findings, apoptotic cells were mainly detected at the tip of the villi, following administration of Gal-1. Moreover, Gal-1-deficient (Lgals1^−/−) mice showed longer villi compared with their wild-type counterparts in vivo. In an experimental model of starvation, fasted wild-type mice displayed reduced villi and lower intestinal weight compared with Lgals1^−/− mutant mice, an effect reflected by changes in the frequency of enterocyte apoptosis. Of note, human small bowel enterocytes were also prone to this pro-apoptotic effect. Thus, Gal-1 is broadly expressed in mucosal tissue and influences the viability of human and mouse enterocytes, an effect which might influence the migration of these cells from the crypt, the integrity of the villus and the epithelial barrier function.     

Myosin IIa activation is crucial in breast cancer derived galectin-1 mediated tolerogenic dendritic cell differentiation

Background

Tolerogenic dendritic cells (tDCs) play important roles in immune tolerance, autoimmune disease, tissue transplantation, and the tumor micro-environment. Factors that induce tDCs have been reported, however the intracellular mechanisms involved are rarely discussed.

Methods

Circulating CD14⁺CD16⁺ of breast cancer patients and induced CD14⁺CD16⁺ DCs were identified as tDCs by treating CD14⁺ monocytes with galectin-1 and cancer cell-derived medium combined with IL-4 and GM-CSF. In addition, the 4T1 breast cancer syngeneic xenograft model was used to investigate the effect of galectin-1 in vivo.

Results

The CD14⁺CD16⁺ tDC population in the breast cancer patients was comparatively higher than that in the healthy donors, and both the MDA-MB-231 conditioned medium and galectin-1 could induce tDC differentiation. In a BALB/c animal model, the 4T1 breast cancer cell line enhanced IL-10 expression in CD11c⁺ DCs which was down-regulated after knocking down the galectin-1 expression of 4T1 cells. Analysis of galectin-1 interacting proteins showed that myosin IIa was a major target of galectin-1 after internalization through a caveolin-dependent endocytosis. Myosin IIa specific inhibitor could diminish the effects of galectin-1 on monocyte-derived tDCs and also block the 4T1 cell induced CD11c⁺/Ly6G⁺/IL-10⁺ in the BALB/c mice.

Conclusions

Galectin-1 can induce tDCs after internalizing into CD14⁺ monocytes through the caveolae-dependent pathway and activating myosin IIa. For the breast cancer patients with a high galectin-1 expression, blebbistatin and genistein show potential in immune modulation and cancer immunotherapy.

General significance

Myosin IIa activation and galectin-1 endocytosis are important in tumor associated tDC development.     

Inhibition of papillomavirus protein function in cervical cancer cells by intrabody targeting

Papillomaviruses (HPVs) are a major cause of human disease, and are responsible for approximately half a million cases of cervical cancer each year. HPVs also cause genital warts, and are the most common sexually transmitted disease in many countries. Despite their importance, there are currently no specific antivirals that are active against HPVs. Papillomavirus protein function is mediated largely by protein-protein interactions, which are difficult to inhibit using conventional approaches. To circumvent these problems, we have prepared an scFv library, and have used this to isolate high-affinity binding molecules that may stearically hinder the association of E6 with p53 and prevent E6-mediated p53 degradation in cervical cancer cells. One of the molecules isolated from the library (GTE6-1), had an affinity for 16E6 of 60nM, and bound within the first zinc finger of the protein. GTE6-1 was able to associate with non-denatured E6 following expression in mammalian cells and could inhibit E6-mediated p53 degradation in in vitro assays. E6-mediated p53 degradation is essential for the continuous growth of cervical cancer cells caused by HPV16. To examine the potential of GTE6-1 as an inhibitor of E6 function in such cells, the molecule was expressed in scFv, diabody and triabody formats in a number of cell lines that are driven to proliferate by the HPV16 oncogenes E6 and E7, including the cervical cancer cell line SiHa. In contrast to small E6-binding peptides containing the ELLG E6-binding motif, GTE6-1 expression lead to changes in nuclear structure, the appearance of apoptosis markers, and an elevation in the levels of p53. No effects were seen with a control scFv molecule, or when GTE6-1 was expressed in cells that are driven to proliferate by simian virus 40 (SV40) T-antigen. Given the accessibility of HPV-associated lesions to topical therapy, our results suggest that large interfering molecules such as intrabodies may be useful inhibitors of viral protein-protein interactions and be particularly appropriate for the treatment of HPV-associated disease.