Natural Compound Shikonin Is a Novel PAK1 Inhibitor and Enhances Efficacy of Chemotherapy against Pancreatic Cancer Cells

Shikonin is the main component of root extracts from the Chinese herbal medicine Lithospermum erythrorhizon, which is commonly used for the treatment of various diseases including cancer. Previous research showed that shikonin suppressed pancreatic cancer growth; nevertheless, its molecular targets and mechanisms have not been elucidated. This study aimed to investigate the interaction and regulatory mechanisms of shikonin on its potential target p21-activated kinase 1 (PAK1). Through a labchip-based screening method, shikonin was identified as a potential bioactive PAK1 inhibitor. Molecular docking technology was used to detect the interaction sites of shikonin and PAK1 kinase. Western blot was performed to validate the mechanism. MTT and flow cytometry were practiced to investigate the effect of shikonin against pancreatic cancer cells. The results show that shikonin significantly inhibited the activity of PAK1 kinase with IC₅₀ value of 7.252 ± 0.054 μM. Molecular docking studies showed that shikonin binds to the ATP-binding pocket of the PAK1 kinase domain. Moreover, shikonin inhibited PAK1 activation and its downstream signaling pathway proteins, while reducing proliferation and inducing apoptosis of pancreatic cancer cells. Further studies showed that the treatment of shikonin sensitized pancreatic cancer cells to chemotherapeutic drugs. These results suggest that shikonin, a potential natural inhibitor targeting PAK1 kinase, has promising potent applications in the treatment of pancreatic cancer and chemotherapy sensitization.     

Regulation of hepatocyte growth factor-mediated urokinase plasminogen activator secretion by MEK/ERK activation in human stomach cancer cell lines

The regulatory mechanisms for the proliferation and the particular invasive phenotypes of stomach cancers are not still fully understood. Up-regulations of hepatocytes growth factor (HGF), its receptor (c-Met), and urokinase-type plasminogen activator (uPA) are correlated with the development and metastasis of cancers. In order to investigate roles of HGF/c-Met signaling in tumor progression and metastasis in stomach cancers, we determined effects of a specific MEK1 inhibitor (PD098059) and a p38 kinase inhibitor (SB203580) on HGF-mediated cell proliferation and uPA expression in stomach cancer cell lines (NUGC-3 and MKN-28). HGF treatment induced the phosphorylations of ERK and p38 kinase in time- and dose- dependent manners. Pre-treatment with PD098059 reduced HGF-mediated cell proliferation and uPA secretion. In contrast, SB203580 pre-treatment enhanced cell proliferation and uPA secretion due to induction of ERK phosphorylation. Stable expression of dominant negative-MEK1 in NUGC-3 cells showed a decrease in HGF-mediated uPA secretion. These results suggest that interaction of a MEK/ERK and a p38 kinase might play an important role in proliferation and invasiveness of stomach cancer cells.     

Krüppel-like factor 4 mediates lysophosphatidic acid-stimulated migration and proliferation of PC3M prostate cancer cells

Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer mortality among men in the United States. Accumulating evidence suggests that lysophosphatidic acid (LPA) serves as an autocrine/paracrine mediator to affect initiation, progression and metastasis of prostate cancer. In the current study, we demonstrate that LPA stimulates migration and proliferation of highly metastatic human prostate cancer, PC-3M-luc-C6 cells. LPA-induced migration of PC-3M-luc-C6 cells was abrogated by pretreatment of PC-3M-luc-C6 cells with the LPA receptor 1/3 inhibitor Ki16425 or small interfering RNA (siRNA)-mediated silencing of endogenous LPA receptor 1, implicating a key role of the LPA-LPA receptor 1 signaling axis in migration of PC-3M-luc-C6 cells. In addition, LPA treatment resulted in augmented expression levels of Krüppel-like factor 4 (KLF4), and siRNA or short-hairpin RNA (shRNA)-mediated silencing of KLF4 expression resulted in the abolishment of LPA-stimulated migration and proliferation of PC-3M-luc-C6 cells. shRNA-mediated silencing of KLF4 expression resulted in the inhibition of in vivo growth of PC-3M-luc-C6 cells in a xenograft transplantation animal model. Taken together, these results suggest a key role of LPA-induced KLF4 expression in cell migration and proliferation of prostate cancer cells in vitro and in vivo.     

PAUF promotes adhesiveness of pancreatic cancer cells by modulating focal adhesion kinase

Pancreatic cancer is a notorious disease with a poor prognosis and low survival rates, which is due to limited advances in understanding of the molecular mechanism and inadequate development of effective treatment options over the decades. In previous studies, we demonstrated that a novel soluble protein named pancreatic adenocarcinoma up-regulated factor (PAUF) acts on tumor and immune cells and plays an important role in metastasis and progression of pancreatic cancer. Here we show that PAUF promotes adhesiveness of pancreatic cancer cells to various extracellular matrix (ECM). Our results further support a positive correlation of activation and expression of focal adhesion kinase (FAK), a key player in tumor cell metastasis and survival, with PAUF expression. PAUF-mediated adhesiveness was significantly attenuated upon blockade of the FAK pathway. Moreover, PAUF appeared to enhance resistance of pancreatic cancer cells to anoikis via modulation of FAK. Our results suggest that PAUF-mediated FAK activation plays an important role in pancreatic cancer progression.     

Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, has shown promising results as a growth inhibitor of HER2-positive cancer cells in vitro. However, similar to other EGFR-targeting drugs, acquired resistance to lapatinib by HER2-positive cancer cells remains a major clinical challenge. To elucidate resistance mechanisms to EGFR/HER2-targeting agents, we performed a systematic quantitative comparison of the phosphoproteome of lapatinib-resistant (LR) human gastric cancer cells (SNU216-LR) versus parental cells (SNU216) using a titanium dioxide (TiO₂) phosphopeptide enrichment method and analysis with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. Biological network analysis of differentially expressed phosphoproteins revealed apparent constitutive activation of the MET-axis phosphatidylinositide 3-kinase (PI3K)/α-serine/threonine-protein kinase (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways in SNU216-LR. Inhibition of the PI3K/AKT and MAPK/ERK signaling pathways in SNU216-LR also leads to cell cycle arrest, confirming the biological network analysis. Lapatinib sensitivity was restored when cells were treated with several molecular targeting agents in combination with lapatinib. Thus, by integrating phosphoproteomic data, protein networks and effects of signaling pathway modulation on cell proliferation, we found that SNU216-LR maintains constitutive activation of the PI3K/AKT and MAPK/ERK pathways in a MET-dependent manner. These findings suggest that pathway activation is a key compensatory intracellular phospho-signaling event that may govern gastric cancer cell resistance to drug treatment.     

Regulation of anti-inflammatory cytokines IL-10 and TGF-β in mouse dendritic cells through treatment with Clonorchis sinensis crude antigen

Dendritic cells (DCs), which are regarded as the most potent antigen-presenting cells, are involved in innate and adaptive immunity. Upon uptake of pathogens, DCs express cell surface markers and secrete cytokines. In this study, we analyzed production of cytokines and found that interleukin (IL)-10 and transforming growth factor (TGF)-β production significantly increased in bone marrow-derived DCs and a mouse DC line, DC2.4, after treatment with crude antigen (CA) from liver fluke, Clonorchis sinensis. However, expression patterns of several activation molecules did not change. In addition, following treatment of DC2.4 cells with antigen from the lung fluke, Paragonimus westermani, production of IL-10 and TGF-β significantly increased compared with groups treated with other parasite antigens, Spirometra erinacei plerocercoid CA and Echinococcus granulosus hydatid cystic fluid. We also found that treatment of DC2.4 cells with C. sinensis CA resulted in rapid and significant phosphorylation of extracellular signal-regulated kinase 1/2, a mitogen-activated protein kinase. Following treatment of DC2.4 cells with C. sinensis CA, treatment with an inhibitor specific to an extracellular signal-regulated kinase inhibited production of IL-10 and TGF-β. Our results suggest that CA from C. sinensis has a role in the anti-inflammatory function of DC cells by inducing IL-10 and TGF-β through activation of extracellular signal-regulated kinase 1/2.     

EGFR and COX-2 Dual Inhibitor: The Design,Synthesis, and Biological Evaluation of Novel Chalcones

For most researchers, discovering new anticancer drugs to avoid the adverse effects of current ones, to improve therapeutic benefits and to reduce resistance is essential. Because the COX-2 enzyme plays an important role in various types of cancer leading to malignancy enhancement, inhibition of apoptosis, and tumor-cell metastasis, an indispensable objective is to design new scaffolds or drugs that possess combined action or dual effect, such as kinase and COX-2 inhibition. The start compounds A1 to A6 were prepared through the diazo coupling of 3-aminoacetophenone with a corresponding phenol and then condensed with two new chalcone series, C7–18. The newly synthesized compounds were assessed against both COX-2 and epidermal growth factor receptor (EGFR) for their inhibitory effect. All novel compounds were screened for cytotoxicity against five cancer cell lines. Compounds C9 and G10 exhibited potent EGFR inhibition with IC₅₀ values of 0.8 and 1.1 µM, respectively. Additionally, they also displayed great COX-2 inhibition with IC₅₀ values of 1.27 and 1.88 µM, respectively. Furthermore, the target compounds were assessed for their cytotoxicity against pancreatic ductal cancer (Panc-1), lung cancer (H-460), human colon cancer (HT-29), human malignant melanoma (A375) and pancreatic cancer (PaCa-2) cell lines. Interestingly, compounds C10 and G12 exhibited the strongest cytotoxic effect against PaCa-2 with average IC₅₀ values of 0.9 and 0.8 µM, respectively. To understand the possible binding modes of the compounds under investigation with the receptor cites of EGFR and COX-2, a virtual docking study was conducted.     

Novel Bradykinin Receptor Inhibitors Inhibit Proliferation and Promote the Apoptosis of Hepatocellular Carcinoma Cells by Inhibiting the ERK Pathway

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Studies have shown that bradykinin (BK) is highly expressed in liver cancer. We designed the novel BK receptor inhibitors J051-71 and J051-105, which reduced the viability of liver cancer cells and inhibited the formation of cancer cell colonies. J051-71 and J051-105 reduced cell proliferation and induced apoptosis in HepG2 and BEL-7402 cells, which may be due to the inhibition of the extracellular regulated protein kinase (ERK) signaling pathway. In addition, these BK receptor inhibitors reversed the cell proliferation induced by BK in HepG2 and BEL-7402 cells by downregulating B1 receptor expression. Inhibiting B1 receptor expression decreased the protein levels of p-ERK and reduced the malignant progression of HCC, providing a potential target for HCC therapy.     

betaig-h3 triggers signaling pathways mediating adhesion and migration of vascular smooth muscle cells through alphavbeta5 integrin

Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis.     

Anti-Cancer Effects of α-Cubebenoate Derived from Schisandra chinensis in CT26 Colon Cancer Cells

α-Cubebenoate derived from Schisandra chinensis has been reported to possess anti-allergic, anti-obesity, and anti-inflammatory effects and to exhibit anti-septic activity, but its anti-cancer effects have not been investigated. To examine the anti-cancer activity of α-cubebenoate, we investigated its effects on the proliferation, apoptosis, and metastasis of CT26 cells. The viabilities of CT26 cells (a murine colorectal carcinoma cell line) and HCT116 cells (a human colon cancer cell line) were remarkably and dose-dependently diminished by α-cubebenoate, whereas the viability of CCD-18Co cells (a normal human fibroblast cell line) were unaffected. Furthermore, α-cubebenoate treatment increased the number of apoptotic CT26 cells as compared with Vehicle-treated cells and increased Bax, Bcl-2, Cas-3, and Cleaved Cas-3 protein levels by activating the MAP kinase signaling pathway. α-Cubebenoate also suppressed CT26 migration by regulating the PI3K/AKT signaling pathway. Furthermore, similar reductions were observed in the expression levels of some migration-related proteins including VEGFA, MMP2, and MMP9. Furthermore, reduced VEGFA expression was found to be accompanied by the phosphorylations of FAK and MLC in the downstream signaling pathway of adhesion protein. The results of the present study provide novel evidence that α-cubebenoate can stimulate apoptosis and inhibit metastasis by regulating the MAPK, PI3K/AKT, and FAK/MLC signaling pathways.     

Design,Synthesis and Biological Evaluation of Novel Pyrazolo[1,2,4]triazolopyrimidine Derivatives as Potential Anticancer Agents

Three novel pyrazolo-[4,3-e][1,2,4]triazolopyrimidine derivatives (1, 2, and 3) were designed, synthesized, and evaluated for their in vitro biological activity. All three compounds exhibited different levels of cytotoxicity against cervical and breast cancer cell lines. However, compound 1 showed the best antiproliferative activity against all tested tumor cell lines, including HCC1937 and HeLa cells, which express high levels of wild-type epidermal growth factor receptor (EGFR). Western blot analyses demonstrated that compound 1 inhibited the activation of EGFR, protein kinase B (Akt), and extracellular signal-regulated kinase (Erk)1/2 in breast and cervical cancer cells at concentrations of 7 and 11 µM, respectively. The results from docking experiments with EGFR suggested the binding of compound 1 at the ATP binding site of EGFR. Furthermore, the crystal structure of compound 3 (7-(4-bromophenyl)-9-(pyridin-4-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine) was determined by single crystal X-ray analysis. Our work represents a promising starting point for the development of a new series of compounds targeting EGFR.     

A naphthalimide-based peptide conjugate for concurrent imaging and apoptosis induction in cancer cells by utilizing endogenous hydrogen sulfide

The excessive production of endogenous hydrogen sulfide (H₂S) in cancer cells leads to enhanced tumor growth and metastasis. On the other hand, decreased endogenous H₂S suppresses tumor growth. The reported approaches for inhibiting tumor growth are selective silencing of the tumor-promoting genes and pharmacological inhibition of these proteins. To enhance the antitumor efficacy of frontline chemotherapeutic agents, herein, we synthesized a highly sensitive endogenous H₂S responsive fluorescent probe, i.e., a hydrogen sulfide-sensing naphthalimide-based peptide conjugate (HSNPc), which showed selective inhibition of proliferation of cancer cells due to apoptosis induction. Furthermore, HSNPc suppressed the glycolytic reserve, a critical energy source for the proliferation of cancer cells. HSNPc also decreased the Young''s modulus of HeLa cells compared to the control cells, which demonstrated a direct relation between cell apoptosis and cell stiffness. Taken together, we demonstrated the dual function of detection and killing of cancer cells by HSNPc that can be likened to a theranostic role.

A hydrogen sulfide sensing naphthalimide based peptide conjugate (HSNPc) worked as a novel cancer cell imaging agent and showed selective cell apoptosis.     

Anti-Cancer Effects of Zotarolimus Combined with 5-Fluorouracil Treatment in HCT-116 Colorectal Cancer-Bearing BALB/c Nude Mice

Zotarolimus is a semi-synthetic derivative of rapamycin and an inhibitor of mammalian target of rapamycin (mTOR) signaling. Currently, zotarolimus is used to prolong the survival time of organ grafts, but it is also a novel immunosuppressive agent with potent anti-proliferative activity. Here, we examine the anti-tumor effect of zotarolimus, alone and in combination with 5-fluorouracil, on HCT-116 colorectal adenocarcinoma cells implanted in BALB/c nude mice. Compared with the control mice, mice treated with zotarolimus or zotarolimus combined with 5-FU showed retarded tumor growth; increased tumor apoptosis through the enhanced expression of cleaved caspase 3 and extracellular signal-regulated kinase (ERK) phosphorylation; reduced inflammation-related factors such as IL-1β, TNF-α, and cyclooxygenase-2 (COX-2) protein; and inhibited metastasis-related factors such as CD44, epidermal growth factor receptor (EGFR), transforming growth factor β (TGF-β), and vascular endothelial growth factor (VEGF). Notably, mice treated with a combination of zotarolimus and 5-FU showed significantly retarded tumor growth, reduced tumor size, and increased tumor inhibition compared with mice treated with 5-FU or zotarolimus alone, indicating a strong synergistic effect. This in vivo study confirms that zotarolimus or zotarolimus combined with 5-FU can be used to retard colorectal adenocarcinoma growth and inhibit tumorigenesis. Our results suggest that zotarolimus may increase the chemo-sensitization of tumor cells. Therefore, zotarolimus alone and zotarolimus combined with 5-FU may be potential anti-tumor agents in the treatment of human colon adenocarcinoma. Future research on zotarolimus may lead to the development of new therapeutic strategies.     

Mechanism of Pterostilbene-Induced Cell Death in HT-29 Colon Cancer Cells

Pterostilbene is a dietary phytochemical that has been found to possess several biological activities, such as antioxidant and anti-inflammatory. Recent studies have shown that it exhibits the hallmark characteristics of an anticancer agent. The aim of the study was to investigate the anticancer activity of pterostilbene against HT-29 human colon cancer cells, focusing on its influence on cell growth, differentiation, and the ability of this stilbene to induce cell death. To clarify the mechanism of pterostilbene activity against colon cancer cells, changes in the expression of several genes and proteins that are directly related to cell proliferation, signal transduction pathways, apoptosis, and autophagy were also evaluated. Cell growth and proliferation of cells exposed to pterostilbene (5–100 µM) were determined by SRB and BRDU assays. Flow cytometric analyses were used for cell cycle progression. Further molecular investigations were performed using quantitative real-time RT-PCR. The expression of the signaling proteins studied was determined by the ELISA method. The results revealed that pterostilbene inhibited proliferation and induced the death of HT-29 colon cancer cells. Pterostilbene, depending on concentration, caused inhibition of proliferation, G1 cell arrest, and/or triggered apoptosis in HT-29 cells. These effects were mediated by the down-regulation of the STAT3 and AKT kinase pathways. It may be concluded that pterostilbene could be considered as a potential therapeutic option in the treatment of colon cancer in the future.     

The Inhibition of Gastric Cancer Cells’ Progression by 23,24-Dihydrocucurbitacin E through Disruption of the Ras/Raf/ERK/MMP9 Signaling Pathway

Gastric cancer is considered to be one of the most common causes of cancer death worldwide due to its high recurrence and metastasis rates. The molecule 23,24-Dihydrocucurbitacin E (DHCE) is a cucurbitacin-derived tetracyclic triterpenoid compound that has anti-tumor activity, but the exact mechanism remains to be elucidated. This research aimed to explore the effects of DHCE on human gastric cancer cells and the possible mechanisms. The results showed that DHCE suppressed proliferation, migration, and invasion of gastric cancer cells, as well as induced apoptosis and G2/M phase arrest. Mechanistically, the potential targets and pathways of DHCE were predicted using database screening and verified using a molecular docking study, fluorescence staining, and Western blot. The results indicated that DHCE obviously inhibited the kinase activity of ERK2 via targeting its ATP-binding domain, destroyed F-actin microfilament, and reduced the expression levels of Ras, p-c-Raf, ERK, p-ERK, and MMP9 proteins. Collectively, our study demonstrated that DHCE suppressed gastric cancer cells’ proliferation, migration, and invasion through targeting ERK2 and disrupting the Ras/Raf/ERK/MMP9 signaling pathway. These properties make DHCE a promising candidate drug for the further design and development of novel and effective Ras/Raf/ERK/MMP9 pathway inhibitors for treating gastric cancer.     

Inhibition of Cytosolic Phospholipase A2α Induces Apoptosis in Multiple Myeloma Cells

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme in releasing arachidonic acid and biosynthesis of its derivative eicosanoids. Thus, the catalytic activity of cPLA2α plays an important role in cellular metabolism in healthy as well as cancer cells. There is mounting evidence suggesting that cPLA2α is an interesting target for cancer treatment; however, it is unclear which cancers are most relevant for further investigation. Here we report the relative expression of cPLA2α in a variety of cancers and cancer cell lines using publicly available datasets. The profiling of a panel of cancer cell lines representing different tissue origins suggests that hematological malignancies are particularly sensitive to the growth inhibitory effect of cPLA2α inhibition. Several hematological cancers and cancer cell lines overexpressed cPLA2α, including multiple myeloma. Multiple myeloma is an incurable hematological cancer of plasma cells in the bone marrow with an emerging requirement of therapeutic approaches. We show here that two cPLA2α inhibitors AVX420 and AVX002, significantly and dose-dependently reduced the viability of multiple myeloma cells and induced apoptosis in vitro. Our findings implicate cPLA2α activity in the survival of multiple myeloma cells and support further studies into cPLA2α as a potential target for treating hematological cancers, including multiple myeloma.     

Phospholipase D inhibitor enhances radiosensitivity of breast cancer cells

Radiation and drug resistance remain the major challenges and causes of mortality in the treatment of locally advanced, recurrent and metastatic breast cancer. Dysregulation of phospholipase D (PLD) has been found in several human cancers and is associated with resistance to anticancer drugs. In the present study, we evaluated the effects of PLD inhibition on cell survival, cell death and DNA damage after exposure to ionizing radiation (IR). Combined IR treatment and PLD inhibition led to an increase in the radiation-induced apoptosis of MDA-MB-231 metastatic breast cancer cells. The selective inhibition of PLD1 and PLD2 led to a significant decrease in the IR-induced colony formation of breast cancer cells. Moreover, PLD inhibition suppressed the radiation-induced activation of extracellular signal-regulated kinase and enhanced the radiation-stimulated phosphorylation of the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase. Furthermore, PLD inhibition, in combination with radiation, was very effective at inducing DNA damage, when compared with radiation alone. Taken together, these results suggest that PLD may be a useful target molecule for the enhancement of the radiotherapy effect.