Exploration of anti-leukemic effect of soft coral-derived 13-acetoxysarcocrassolide: Induction of apoptosis via oxidative stress as a potent inhibitor of heat shock protein 90 and topoisomerase II.

13-Acetoxysarcocrassolide (13-AC) is a marine cembranoid derived from the aquaculture soft coral of Lobophytum crassum. The cytotoxic effect of 13-AC against leukemia cells was previously reported but its mechanism of action is still unexplored. In the current study, we showed that 13-AC induced apoptosis of human acute lymphoblastic leukemia Molt4 cells, as evidenced by the cleavage of PARP and caspases, phosphatidylserine externalization, as well as the disruption of mitochondrial membrane potential. The use of N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, attenuated the cytotoxic effect induced by 13-AC. Molecular docking and thermal shift assay indicated that the cytotoxic mechanism of action of 13-AC involved the inhibition of heat shock protein 90 (Hsp 90) activity by eliciting the level of Hsp 70 and topoisomerase IIα in Molt4 cells. 13-AC also exhibited potent antitumor activity by reducing the tumor volume (48.3%) and weight (72.5%) in the in vivo Molt4 xenograft mice model. Our findings suggested that the marine cembranoid, 13-AC, acted as a dual inhibitor of Hsp 90 and topoisomerase IIα, exerting more potent apoptotic activity via the enhancement of ROS generation.

Novel 9-Benzylaminoacridine Derivatives as Dual Inhibitors of Phosphodiesterase 5 and Topoisomerase II for the Treatment of Colon Cancer.

It has been shown that phosphodiesterase 5 (PDE5) inhibitors have anticancer effects in a variety of malignancies in both in vivo and in vitro experiments. The role of cGMP elevation in colorectal carcinoma (CRC) has been extensively studied. Additionally, DNA topoisomerase II (Topo II) inhibition is a well-established mechanism of action that mediates the effects of several approved anticancer drugs such as doxorubicin and mitoxantrone. Herein, we present 9-benzylaminoacridine derivatives as dual inhibitors of the PDE5 and Topo II enzymes. We synthesized 31 derivatives and evaluated them against PDE5, whereby 22 compounds showed micromolar or sub-micromolar inhibition. The anticancer activity of the compounds was evaluated with the NCI 60-cell line testing. Moreover, the effects of the compounds on HCT-116 colorectal carcinoma (CRC) were extensively studied, and potent compounds against HCT-116 cells were studied for their effects on Topo II, cell cycle progression, and apoptosis. In addition to exhibiting significant growth inhibition against HCT116 cells, compounds 11, 12, and 28 also exhibited the most superior Topo II inhibitory activity and low micromolar PDE5 inhibition and affected cell cycle progression. Knowing that compounds that combat cancer through multiple mechanisms are among the best candidates for effective therapy, we believe that the current class of compounds merits further optimization and investigation to unleash their full therapeutic potential.

Metformin Enhancement of Therapeutic Effects of 5-Fluorouracil and Oxaliplatin in Colon Cancer Cells and Nude Mice.

Studies have demonstrated that metformin has antitumor effects in addition to therapeutic effects on hyperglycemia; however, few studies have explored the effects of metformin in chemotherapy. Therefore, we hypothesized that the administration of metformin would enhance the therapeutic effects of 5-fluorouracil and oxaliplatin (FuOx) to inhibit the growth of colorectal cancer (CRC) cells in vitro and in vivo. The results of our in vitro experiments demonstrated that metformin significantly increased the effects of FuOx with respect to cell proliferation (p < 0.05), colony formation (p < 0.05), and migration (p < 0.01) and induced cell cycle arrest in the G0/G1 phase in HT29 cells and the S phase in SW480 and SW620 cells (p < 0.05). Flow cytometry analysis revealed that metformin combined with FuOx induced late apoptosis (p < 0.05) by mediating mitochondria-related Mcl-1 and Bim protein expression. Furthermore, in vivo, metformin combined with FuOx more notably reduced tumor volume than FuOx or metformin alone did in BALB/c mice (p < 0.05). These findings demonstrate that metformin may act as an adjunctive agent to enhance the chemosensitivity of CRC cells to FuOx. However, further clinical trials are warranted to validate the clinical implications of the findings.

Genomic and Metabolomic Landscape of Right-Sided and Left-Sided Colorectal Cancer: Potential Preventive Biomarkers.

Colorectal cancer (CRC) is the third most common cancer worldwide. The incidence and mortality rates of CRC are significantly higher in Taiwan than in other developed countries. Genes involved in CRC tumorigenesis differ depending on whether the tumor occurs on the left or right side of the colon, and genomic analysis is a keystone in the study and treatment of CRC subtypes. However, few studies have focused on the genetic landscape of Taiwanese patients with CRC. This study comprehensively analyzed the genomes of 141 Taiwanese patients with CRC through whole-exome sequencing. Significant genomic differences related to the site of CRC development were observed. Blood metabolomic profiling and polygenic risk score analysis were performed to identify potential biomarkers for the early identification and prevention of CRC in the Taiwanese population. Our findings provide vital clues for establishing population-specific treatments and health policies for CRC prevention in Taiwan.

EGFR expression in patients with stage III colorectal cancer after adjuvant chemotherapy and on cancer cell function.

The epidermal growth factor receptor (EGFR)/RAS/RAF/MEK/MAPK pathway plays a crucial role in the carcinogenesis, invasion and metastasis of colorectal cancer (CRC). However, its role in the prognosis and prediction of relapse in patients with stage III CRC after adjuvant chemotherapy remains controversial. In the present study, the clinicopathological features of 173 patients with stage III CRC who underwent radical resection and adjuvant chemotherapy with the fluoropyrimidine/folinic acid, and oxaliplatin (FOLFOX) regimen, and their prognostic values of EGFR expression were retrospectively analyzed. By conducting an in vitro CRC cell line study through the knockdown of EGFR expression, we analyzed cell proliferation, colony formation and migration. Positive EGFR expression and an abnormal postoperative serum carcinoembryonic antigen (CEA) level were found to be significant independent negative predictive factors for postoperative relapse. Furthermore, positive EGFR expression was a significant independent negative prognostic factor for disease-free survival (DFS) and overall survival (OS). Additionally, an in vitro cell line study showed that the knockdown of EGFR expression significantly reduced CRC cell proliferation, colony formation and migration. The results of in vitro and in vivo experiments demonstrated that EGFR expression had a prognostic value for OS and DFS, as well as predictive roles for postoperative relapse, in patients with stage III CRC. By analyzing both EGFR expression and the postoperative CEA, the patients with stage III CRC who were at a high risk of postoperative relapse, or mortality following adjuvant chemotherapy could be identified. In short, CRC cells with EGFR expression would exhibit a highly malignant behavior.

Role of Exogenous Hsp72 on Liver Dysfunction during Sepsis.

This study examined the role of exogenous heat shock protein 72 (Hsp72) in reversing sepsis-induced liver dysfunction. Sepsis was induced by cecal ligation and puncture. Liver function was determined on the basis of the enzymatic activities of serum glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). Apoptosis was determined using terminal deoxynucleotidyl transferase dUTP nick end labeling staining. B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3 and caspase-9, and cleaved poly (ADP-ribose) polymerase (PARP) protein expressions were analyzed using Western blotting. Results showed GOT and GPT levels increased during sepsis, and levels were restored following the administration of human recombinant Hsp72 (rhHsp72). Increased liver tissue apoptosis was observed during sepsis, and normal apoptosis resumed on rhHsp72 administration. The Bcl-2/Bax ratio, cleaved caspase-3, caspase-9, and PARP protein expressions in the liver tissues were upregulated during sepsis and normalized after rhHsp72 treatment. We conclude that, during sepsis, exogenous Hsp72 restored liver dysfunction by inhibiting apoptosis via the mitochondria-initiated caspase pathway.

Release of endogenous heat shock protein 72 on the survival of sepsis in rats.

BACKGROUND: This study was undertaken to clarify the role of extracellular heat shock protein 72 on the survival of sepsis and to determine possible factor(s) that may be responsible for it. MATERIALS AND METHODS: Sepsis was induced by cecal ligation and puncture. Changes in serum levels of heat shock protein (Hsp72) and cytokines were determined during sepsis, and the results were correlated with the survival. Effects of heat pretreatment on Hsp72 expression in septic rat leukocytes and those of septic rat serum, lipopolysaccharide (LPS), and certain cytokines on the release of Hsp72 in macrophage NR8383 cells were determined. RESULTS: Circulating Hsp72 levels were increased during the progress of sepsis (0, 5.5, 6.5, 10, and 6.5 ng/mL at 0, 3, 6, 9, and 18 h after cecal ligation and puncture, respectively) and the increases were correlated positively with survival rates. LPS triggered the release of Hsp72 in heat pretreated animals. Heat pretreatment increased Hsp72 expression in nonsepsis (+535%, P < 0.01) and sepsis (+116%, P<0.01%) rat leukocytes. Incubation of sepsis rat serum with NR8383 cells increased levels of extracellular heat shock protein 72 in cultured medium. Cytokine profiling revealed that among the 19 cytokines screened, four of them were increased as follows: cytokine-induced neutrophil chemoattractant 3 (+211.3%, P < 0.05), interleukin 10 (+147%, P < 0.05), MCP-1 (+49.6%, P < 0.05), and tumor necrosis factor alpha (+51.8%, P < 0.05). MCP-1 and LPS were capable of releasing Hsp72 from NR8383 cells. CONCLUSIONS: These results demonstrate that the increases in the levels of circulating Hsp72 had a beneficial effect in improving animal survival during the progress of sepsis. The increases in circulating Hsp72 may be mediated via MCP-1 and/or LPS.

Nonlethal dose of silver nanoparticles attenuates TNF-α-induced hepatic epithelial cell death through HSP70 overexpression.

Silver nanoparticles (Ag-nps) have been widely used in various biomedical products. Compared with its hazardous effects extensively being studied, rare attention has been paid to the potential protective effect of Ag-nps to human health. The present study was designed to evaluate the protective effects of Ag-nps and heat shock treatment on tumor necrosis factor-α (TNF-α)-induced cell damage in Clone 9 cells. Clone 9 cells were pretreated with nonlethal concentration of Ag-nps (1 µg/ml) or heat shock, and then cell damages were induced by TNF-α (1 ng/ml). Protective effects of Ag-nps administration or heat shock treatment were determined by examining the TNF-α-induced changes in cell viabilities. The results showed that the intensity of cytotoxicity produced by TNF-α was alleviated upon treatment with nonlethal concentration of Ag-nps (1 µg/ml). Similar protective effects were also found upon heat shock treatment. These data demonstrate that Ag-nps and heat shock treatment were equally capable of inducing heat shock protein 70 (HSP70) protein expression in Clone 9 cells. The results suggest that clinically Ag-nps administration is a viable strategy to induce endogenous HSP70 expression instead of applying heat shock. In conclusion, our study for the first time provides evidence that Ag-nps may act as a viable alternative for HSP70 induction clinically.

Effect of carbon dioxide inhalation on pulmonary hypertension induced by increased blood flow and hypoxia.

There is now increasing evidence from the experimental and clinical setting that therapeutic hypercapnia from intentionally inspired carbon dioxide (CO(2)) or lower tidal volume might be a beneficial adjunct to the strategies of mechanical ventilation in critical illness. Although previous reports indicate that CO(2) exerts a beneficial effect in the lungs, the pulmonary vascular response to hypercapnia under various conditions remains to be clarified. The purpose of the present study is to characterize the pulmonary vascular response to CO(2) under the different conditions of pulmonary hypertension secondary to increased pulmonary blood flow and secondary to hypoxic pulmonary vasoconstriction. Isolated rat lung (n = 32) was used to study (1) the vasoactive action of 5% CO(2) in either N(2) (hypoxic-hypercapnia) or air (normoxic-hypercapnia) at different pulmonary arterial pressure levels induced by graded speed of perfusion flow and (2) the role of nitric oxide (NO) in mediating the pulmonary vascular response to hypercapnia, hypoxia, and flow-associated pulmonary hypertension. The results indicated that inhaled CO(2) reversed pulmonary hypertension induced by hypoxia but not by flow alteration. Endogenous NO attenuates hypoxic pulmonary vasoconstriction but does not augment the CO(2)-induced vasodilatation. Acute change in blood flow does not alter the endogenous NO production.

Suppression of mitochondrial ATPase inhibitor protein (IF1) in the liver of late septic rats.

Sepsis and ensuing multiple organ failure continue to be the most leading cause of death in critically ill patients. Despite hepatocyte-related dysfunctions such as necrosis, apoptosis as well as mitochondrial damage are observed in the process of sepsis, the molecular mechanism of pathogenesis remains uncertain. We recently identified one of the differentially expressed genes, mitochondrial ATPase inhibitor protein (IF1) which is down-regulated in late septic liver. Hence, we further hypothesized that the variation of IF1 protein may be one of the causal events of the hepatic dysfunction during late sepsis. The results showed that the elevated mitochondrial F0F1-ATPase activity is concomitant with the decline of intramitochondrial ATP concentration in late septic liver. In addition, the key finding of this study showed that the mRNA and the mitochondrial content of IF1 were decreased in late sepsis while no detectable IF1 was found in cytoplasm. When analyzed by immunoprecipitation, it seems reasonable to imply that the association capability of IF1 with F1-ATPase beta-subunit is not affected. These results confirm the first evidence showing that the suppression of IF1 expression and subsequent elevated mitochondrial F0F1-ATPase activity might contribute to the bioenergetic failure in the liver during late sepsis.

Down-regulation of N-methyl D-aspartate receptor in rat-modeled disuse osteopenia.

Lack of mechanical stress may result in osteoporosis; however, the underlying mechanisms of disuse osteoporosis remain unclear. It has been indicated that mechanical loading causes extracellular glutamate accumulation in osteoblasts. We hypothesized that the glutamate receptor mediation on bone cells might also be involved in mechanically stimulated osteogenesis. In this study, we investigated the changes of bone formation and the expressions of osteogenic genes and N-methyl D-aspartate (NMDA) receptors, the major glutamate receptors, in disused bones. Rat modeled disuse osteopenia in hind limbs was induced by a 3-week tail suspension in Sprague-Dawley rats. Bone mineral density and trabecular bone volume of distal femurs were measured to verify the osteopenia of disused bones. The mRNA expressions of cbfa1/Runx2, type I collagen, alkaline phosphatase (ALP) and osteocalcin (OC) in bones were measured as osteogenic markers. The influences of mechanical unloading on the expressions of NMDA receptors (NR1 and NR2D) in bones were also examined. The effects of NMDA mediation on osteogenesis were tested by a treatment of MK-801, a non-competitive NMDA receptor antagonist, in cultured osteoblasts and bone marrow stroma cells. Our result showed that mRNA expressions of cbfa1/Runx2, type I collagen, ALP and OC were significantly decreased in disused bones. The mRNA and protein expressions of NR1 and NR2D were significantly decreased in disused bones; furthermore, immunolocalization of both receptors showed decreases in osteoblasts, but not in osteoclasts. The results from the in vitro study showed that MK-801 inhibited mRNA expression of cbfa1/Runx2 in bone marrow stroma cells and also inhibited those of collagen type I, ALP and OC of osteoblasts in a dose-dependent manner. These results suggest that NMDA receptor mediation may play an important role in transmitting mechanical loading in bones, and decreases of the expressions of NMDA receptors in disused bones, especially in osteoblasts, may contribute to the decrease of osteogenesis.