结直肠癌微环境诱导腹膜间皮细胞凋亡的体外实验研究

目的:创建发生腹膜转移早期时结直肠癌对人腹膜间皮细胞HMrSV5的损伤模型,探讨结直肠癌细胞对腹膜间皮细胞的影响及凋亡相关蛋白的表达。方法:将人结直肠癌细胞培养液上清加入到人腹膜间皮细胞系HMrSV5。共培养后,光镜下观察间皮细胞的形态变化、CCK-8检测增殖改变、流式细胞仪判断凋亡比例、Western-blot检测凋亡相关蛋白Bcl-2和Bax、Transwell小室检测受损间皮细胞对结直肠癌细胞的影响。结果:结直肠癌细胞培养液上清接触间皮细胞后,发生了形态改变,细胞的增殖受到了抑制,凋亡蛋白Bcl-2与Bax表达失调。同时,受损间皮能够促进结直肠癌细胞的迁移。结论:结直肠癌细胞在发生腹膜转移的早期时候,就可以通过其分泌物促使间皮细胞发生凋亡,凋亡的发生与Bcl-2和Bax的失调有关;同时受损间皮能够反作用于结直肠癌细胞,促进其迁移。我们从中意识到,保护间皮细胞免受损伤,在预防和治疗结直肠癌腹膜转移中或许能起到一定的作用。     

The apoptotic effect of apigenin on human gastric carcinoma cells through mitochondrial signal pathway

This study aims to explore the apoptotic function of apigenin on the gastric cancer cells and the related mechanism. The gastric cancer cell lines HGC-27 and SGC-7901, and normal gastric epithelial cell line GES1 were treated with different concentrations of apigenin. Cell proliferation was tested. Morphological changes of the apoptotic cells were observed after Hoechst33342 staining. The apoptosis rate of the gastric cancer cells were measured with flow cytometry. Changes of the cell cycle were explored. The mitochondrial membrane potential changes were analyzed after JC-1 staining. Bcl-2 family proteins and caspases-3 expression with apigenin treatment was analyzed by real-time PCR. Cell proliferation of HGC-27 and SGC-7901 was inhibited by apigenin, and the inhibition was dose-time-dependent. Gastric carcinoma cells treated by apigenin had no obvious cell cycle arrest, but were observed with the higher apoptosis rate and the typical apoptotic morphological changes of the cell nucleus. JC-1 staining showed that apigenin could reduce mitochondrial membrane potential of gastric carcinoma cells. Real-time PCR results showed that apigenin significantly increased caspase-3 and Bax expression level, and down-regulated Bcl-2 expression in a dose-dependent manner in gastric carcinoma cells. However, the GES1 was almost not affected by apigenin treatment. Apigenin can inhibit cell lines HGC-27 and SGC-7901 proliferation in a time and dose-dependent manner, reduce anti-apoptotic protein Bcl-2 levels, enhance apoptosis-promoting protein Bax level, result in mitochondrial membrane potential decreasing and caspase-3 enzyme activating, then lead to cell apoptosis.     

Anticancer bioactive peptide (ACBP) inhibits gastric cancer cells by upregulating growth arrest and DNA damage-inducible gene 45A (GADD45A)

Recently, we reported that anticancer bioactive peptide (ACBP), purified from goat spleens immunized with human gastric cancer extracts, significantly inhibited gastric cancer cells in vitro and gastric tumors in vivo via repressing cell growth and promoting apoptosis, making it a promising potential biological anticancer drug. However, it is not known what genes are functionally required for the ACBP effects. Here, we first found that two tumor suppressor genes, cyclin-dependent kinase inhibitor 2B (CDKN2B) and growth arrest and DNA damage-inducible alpha (GADD45A), were upregulated significantly in the cells with ACBP treatment by microarray screening and the findings were validated by real-time RT-PCR. Next, GADD45A mRNA and protein expressions were downregulated in the gastric cancer cells by lentivirus-mediated RNAi; then, cell viability, cell cycle, and apoptosis were assayed by MTT and flow cytometry. Interestingly, our results indicated that cell viability was not dependent on GADD45A without ACBP treatment; however, cell sensitivity to ACBP was significantly decreased in ACBP-treated gastric cancer cells with GADD45A downregulation. Therefore, we demonstrate that GADD45A was functionally required for ACBP to inhibit gastric cancer cells, suggesting that GADD45A may become a biomarker for ACBP sensitivity. Our findings have significant implications on the molecular mechanism understanding, biomarker development, and anticancer drug development of ACBP.     

Induction of gastric cancer cell adhesion through transforming growth factor-beta1-mediated peritoneal fibrosis

Background

Peritoneal dissemination is one of the main causes of death in gastric cancer patients. Transforming growth factor-beta1 (TGF-β1), one of the most potent fibrotic stimuli for mesothelial cells, may play a key role in this processing. The purpose of this study is to elucidate the effects of TGF-β1 on regulation of gastric cancer adhesion to mesothelial cells.

Methods

Peritoneal tissues and peritoneal wash fluid were obtained for hematoxylin and eosin staining or ELISA to measure fibrosis and TGF-β1 levels, respectively. The peritoneal mesothelial cell line, HMrSV5, was used to determine the role of TGF-β1 in regulation of gastric cancer cell adhesion to mesothelial cells and expression of collagen, fibronectin, and Smad 2/3 by using adhesion assay, western blot, and RT-PCR.

Results

The data showed that TGF-β1 treatment was able to induce collagen III and fibronectin expression in the mesothelial cells, which was associated with an increased adhesion ability of gastric cancer cells, but knockdown of minimal sites of cell binding domain of extracellular matrix can partially inhibit these effects.

Conclusion

Peritoneal fibrosis induced by TGF-β1 may provide a favorable environment for the dissemination of gastric cancer.     

乳斑肿瘤相关巨噬细胞诱导人腹膜间皮HMR-sv5细胞损伤的机制研究

  目的   研究胃癌细胞对巨噬细胞的诱导作用, 分析活化巨噬细胞对间皮细胞的损伤作用及机制。   目法   人正常胃腺上皮细胞系GES-1及人低分化胃癌细胞系SGC-7901与人单核巨噬细胞THP-1共培养, 诱导后者分化, 研究后者对人腹膜间皮细胞HMR-sv5的损伤作用及分子机制。   结果   胃癌细胞诱导THP-1形成肿瘤相关巨噬细胞(TAM), M1型巨噬细胞表面抗原的表达显著下调, 而M2表型的表面抗原明显上调, 细胞形态也发生明显改变。TAM显著抑制正常间皮细胞生长, 促进间皮细胞凋亡和上皮间质转化。   结论   胃癌细胞诱导巨噬细胞发生表型和功能转化, 进而导致间皮细胞发生EMT和凋亡, 促进形成腹膜转移癌。      

Hepatocyte growth factor (HGF) produced by peritoneal fibroblasts may affect mesothelial cell morphology and promote peritoneal dissemination

Mesothelial cell monolayers have been reported to prevent infiltration of cancer cells into the peritoneum. We have previously reported that peritoneal fibrosis induced by gastric cancer cells prior to metastatization may provide a congenial environment for peritoneal metastases. In this study, we investigated the effects of peritoneal fibroblasts on peritoneal mesothelial cell morphology. Human gastric cancer (OCUM-2MD3), peritoneal fibroblast (NF-2P) and mesothelial (MS-1) cell lines were established in our laboratory. Histology of the peritoneum was investigated following intraperitoneal inoculation of serum-free conditioned media (SF-CM) from OCUM-2MD3 cells into nude mice. SF-CM from peritoneal fibroblasts was added to monolayer-cultured mesothelial cells, and their morphology was examined by phase-contrast microscopy. This experiment was conducted in the presence and absence of neutralizing antibodies against various factors. Mesothelial cells exposed to fibroblasts proliferation became hemispherical and separated from each other, while unexposed mesothelium remained as a flat monolayer. Cultured-mesothelial cells rounded up or exhibited a fibroblast-like shape following the addition of peritoneal fibroblast SF-CM. Anti-hepatocyte growth factor (HGF) neutralizing antibody partly inhibited this effect. We suggest that soluble factors, such as HGF, produced by peritoneal fibroblasts affect the morphology of mesothelial cells in monolayers so that the resulting environment may become prone to the peritoneal dissemination of cancer cells. © 1996 Wiley-Liss, Inc.     

Adhesion of human gastric and pancreatic cancer cells to peritoneal mesothelial cells is mediated by CD44 and beta(1) integrin

Peritoneal dissemination is a common cause of the recurrence of gastric or pancreatic cancer after patients have undergone surgery. The presence of peritoneal metastasis after surgery affects the prognosis of patients with gastric or pancreatic cancer. Very little is known about the biochemical processes involved in the initial attachment of cancer cells to peritoneal mesothelial cells. We conducted in vitro and in vivo studies to assess the role of adhesion molecules in this process, using 5 cell lines derived from human gastric and pancreatic cancers. NUGC-4 and SW1990 cells, which disseminate earlier than the other 3 types of cancer cells after inoculation into the abdominal cavity of nude mice, express large amounts of CD44H. We found that NUGC-4 and SW1990 cells adhere to monolayers of mesothelial cells more firmly than the other cell lines, as shown by adhesion assays performed at 4 degrees C. The adhesion of NUGC-4 and SW1990 cells to mesothelial cells was partially inhibited by antibodies against CD44H or the beta(1) subunit of integrin, and they almost completely blocked adhesion when these 2 antibodies were used in combination in vitro. These 2 antibodies also inhibited the peritoneal metastasis of NUGC-4 and SW1990 cells and prolonged their mean survival time in vivo. These findings suggest that CD44H and beta(1) integrin play important roles in the initial attachment of gastric and pancreatic cancer cells to mesothelial cells. Our results suggest that changes in the expression of CD44H and beta(1) integrin in cancer cells is associated with their ability to adhere to peritoneal mesothelial cells, and thus with the peritoneal metastatic ability of gastric and pancreatic cancer cells. Therefore, the expression of CD44H and beta(1) integrin in gastric and pancreatic cancers could be used as prognostic indicators of peritoneal metastasis. It is possible that a treatment strategy that interferes with the functions of CD44H or beta(1) integrin may result in decreased intra-abdominal spread of cancer.     

胃癌细胞与腹膜间皮相互作进用促腹膜纤维化致腹膜转移的研究

  目的   研究间皮细胞对胃癌细胞的抵御作用, 模拟游离癌细胞在接触腹膜前通过其分泌物导致的腹膜增厚、纤维化的过程, 同时观察了间皮细胞对胃癌细胞迁移及侵袭能力的反作用。   方法  用荧光显微镜观察共培养时间皮细胞对胃癌细胞的抵御作用; 体内实验观察腹膜变化; 电镜、光镜下观察体外实验中间皮层在胃癌-腹膜相互作用中的损伤和细胞骨架变化; 采用Millicell小室共培养观察间皮细胞对胃癌细胞迁移及侵袭能力的反作用。   结果  正常间皮细胞可防止肿瘤细胞对于腹膜的粘附, 受损脱落后胃癌细胞可轻易粘附。体内外实验均显示接触胃癌细胞上清后腹膜间皮细胞受损、凋亡, 并且受损残余的间皮可以反作用于癌细胞, 使其迁移转移力提高。   结论  正常间皮细胞可以抵御胃癌细胞的侵袭, 受损伤刺激后的间皮细胞可以反作用于胃癌细胞促进其迁移及侵袭。      

Transforming growth factor-beta1 signaling blockade attenuates gastric cancer cell-induced peritoneal mesothelial cell fibrosis and alleviates peritoneal dissemination both in vitro and in vivo

Peritoneal dissemination is the most frequent metastatic pattern of advanced gastric cancer and the main cause of death in gastric cancer patients. Transforming growth factor-beta1 (TGF- ß1), one of the most potent fibrotic stimuli for human peritoneal mesothelial cells, has been shown to play an important role in this process. In this study, we investigated the effect of TGF- ß1 signaling blockade in gastric cancer cell (GCC)-induced human peritoneal mesothelial cell (HPMC) fibrosis. HPMCs were cocultured with the high TGF- ß1 expressing GCC line SGC-7901 and various TGF- ß1 signaling inhibitors or SGC-7901 transfected with TGF-ß1-specific siRNA. HPMC fibrosis was monitored on the basis of morphology. Expression of the epithelial cell marker, E-cadherin, and the mesenchymal marker, α-smooth muscle actin (α-SMA), was evaluated by Western blotting and immunofluorescence confocal imaging. GCC adhesion to HPMC was also assayed. In nude mouse tumor model, the peritoneal fibrotic status was monitored by immunofluorescent confocal imaging and Masson’s trichrome staining; formation of metastatic nodular and ascites fluid was also evaluated. Our study demonstrated that GCC expressing high levels of TGF-ß1 induced HMPC fibrosis, which is characterized by both upregulation of E-cadherin and downregulation of α-SMA. Furthermore, HPMC monolayers fibrosis was reversed by TGF- ß1 signaling blockade. In vivo, the TGF- ß1 receptor inhibitor SB-431542 partially attenuated early-stage gastric cancer peritoneal dissemination (GCPD). In conclusion, our study confirms the significance of TGFß1 signaling blockade in attenuating GCPD and may provide a therapeutic target for clinical therapy.     

Tumor-associated mesothelial cells are negative prognostic factors in gastric cancer and promote peritoneal dissemination of adherent gastric cancer cells by chemotaxis

Peritoneal dissemination is highly frequent in gastric cancer. Damage to human peritoneal mesothelial cell (HPMC) barriers provokes gastric cancer peritoneal dissemination (GCPD), the key events during GCPD, is characterized by fibroblastic development. In this study, we have studied the association between fibroblast activation protein (FAP) expression in peritoneum and the pathological features of the primary tumor. The clinical prognosis of gastric cancer patients was evaluated according to FAP expression. In a gastric cancer cell-HPMC co-culture system, expression of E-cadherin, α-smooth muscle actin, and FAP were evaluated by Western blotting. Gastric cancer cell migration and adhesion to HPMC were also assayed. Our results showed positive peritoneal staining of FAP in 36/86 cases (41.9 %), which was associated with a higher TNM stage in primary gastric cancer and higher incidence of GCPD (both p?<?0.05). Survival analysis showed FAP expression was an independent prognostic factor of poor survival (p?=?0.02). Peritoneum of FAP-positive expression exhibited a distinct fibrotic development and expressed higher level of the mesenchymal marker α-SMA, which was confirmed by the in vitro Western blot assay. In HPMC and gastric cancer cell adherence assay, SGC-7901 cells preferentially adhered to TA-HPMC at different cell densities (both p?<?0.05). Additionally, SGC-7901 cells were more prone to chemotaxis by FAP-expressed tumor-associated–human peritoneal mesothelial cells (TA-HPMC) compared with HPMC co-cultured with normal gastric glandular epithelial cells in a time-dependent manner (both p?<?0.05). Our study indicated a positive correlation between peritoneum FAP expression and GCPD. FAP-expressed TA-HPMC might be an important cellular component and instigator of GCPD.     

Tumour-induced apoptosis in human mesothelial cells: a mechanism of peritoneal invasion by Fas Ligand/Fas interaction

Gastrointestinal carcinomas frequently disseminate within the abdominal cavity to form secondary peritoneal metastases. Invasion of the peritoneal mesothelium is fundamental to this process, yet the underlying invasive mechanisms remain unclear. Preliminary in vitro work suggested that tumour cells can induce mesothelial apoptosis, representing a novel mechanism of peritoneal invasion. We examined the role of tumour cell-induced mesothelial apoptosis and explored the role of the death ligand/receptor system, Fas Ligand/Fas, as mediators of the apoptotic process. Cultured human mesothelial cells were used to establish in vitro co-culture models with the SW480 colonic cancer cell line. Tumour-induced mesothelial apoptosis was confirmed by phase-contrast microscopy and apoptotic detection assays. Human mesothelial cells and SW480 tumour cells constitutively expressed Fas and Fas Ligand mRNA and protein as determined by RT-PCR and confocal fluorescent microscopy. Stimulation of human mesothelial cells with anti-Fas monoclonal antibody or crosslinked soluble Fas Ligand-induced apoptosis, confirming the functional status of the Fas receptor. Pretreatment of SW480 cells with a blocking recombinant anti-Fas Ligand monoclonal antibody significantly reduced mesothelial apoptosis, indicating that tumour-induced mesothelial apoptosis may, in part, be mediated via a Fas-dependent mechanism. This represents a novel mechanism of mesothelial invasion and offers several new targets for therapeutic intervention.     

Adhesion molecules and TGF-β1 are involved in the peritoneal dissemination of NUGC-4 human gastric cancer cells

Peritoneal dissemination frequently occurs after surgery in patients with gastric cancer. The presence of peritoneal metastasis after surgery affects prognosis. Very little is known about the biochemical processes involved in the initial attachment of gastric cancer cells to peritoneal mesothelial cells. We conducted in vitro and in vivo studies to assess the role of adhesion molecules and TGF-β1 in this process, using 4 cell lines derived from human gastric cancers. NUGC-4 cells, which disseminate early after inoculation into the abdominal cavity of nude mice, predominantly express CD44H and β₁ integrin. We found that NUGC-4 cells adhered to monolayers of mesothelial cells more firmly than to other cell lines. Adhesion of NUGC-4 cells to mesothelial cells was partially inhibited by antibodies against CD44H or the β₁ subunit of integrin and was completely blocked by a combination of these 2 antibodies. Treatment with ligands for CD44H and β₁ integrin also inhibited adhesion. In the NUGC-4 cell culture medium, larger amounts of TGF-β₁ were detected in relation to the increase in cancer cells than in the other cell lines. TGF-β1 increased the expression of CD44H in NUGC-4 cells and in mesothelial cells and augmented adhesion and implantation of NUGC-4 cells to mesothelial cells accompanied by accumulation of extracellular matrix (ECM) components. Treatment with antibodies against both CD44H and β₁ integrin inhibited the dissemination of NUGC-4 cells in the peritoneal cavity of nude mice and prolonged their survival time. Our findings suggest that CD44H and integrins mediate the initial attachment of gastric cancer cells to mesothelial cells and that TGF-β1 participates in the promotion of the disease. Increased expression of CD44H and of the amount of ligands for CD44H and integrins induced by TGF-β1 promotes early development of peritoneal dissemination. Int. J. Cancer 70:612–618. © 1997 Wiley-Liss Inc     

Purification of a Pd20–TNFα fusion protein that prevents liver metastasis of gastric cancer

The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human tumour necrosis factor (TNF) α, and a prokaryotic expression vector was established to express the pd20–TNFα fusion protein. After purification and identification, the preventive effects of the fusion protein on liver metastasis of gastric cancer were observed in mice. The whole gene synthesis method was used for pd20–TNFα fusion gene preparation, and a pd20–TNFα prokaryotic expression vector was constructed. The vector was induced and expressed in Escherichia coli BL21. The expression products were analysed and verified by SDS-PAGE electrophoresis and Western blot analysis. The Ni-NTA column method was used to purify the fusion protein, and the L929 cytotoxicity method was used to detect biological activity. Flow cytometry apoptosis experiments and invasion assays were performed to observe the effects of the fusion protein on apoptosis and metastasis of gastric cancer cells with high potential for liver metastasis. Thirty nude mice with liver metastasis of gastric cancer were established and then randomly divided into three groups of ten mice each. The Pd20–TNFα recombinant protein (1.2?×?10⁶ U/kg day) or standard TNFα (1.2?×?10⁶ U/kg day) saline was administered via tail vein injection for 7 consecutive days. The pathological changes in various organs of nude mice were observed 4 weeks later. The size of the gastric cancer, the incidence of liver metastasis and the number of liver metastases were measured and calculated. We successfully constructed a Pd20–TNFα recombinant plasmid and prepared the fusion protein. Detection of the pd20–TNFα protein by immunofluorescence showed a very strong expression in liver tissue, suggesting a targeting of the fusion protein to the liver. The L929 cytotoxicity assays showed that the pd20–TNFα fusion purified protein had a significant lethal effect on L929 cells, with a killing activity of up to 7.6?×?10⁶ IU/ml. The apoptosis experiments showed that as the concentration of the fusion protein increased, the early gastric cancer cell apoptosis also increased, with the early apoptosis rate increasing from 5.99 % to 9.04 %. Cell invasion experiments showed that the purified pd20–TNFα fusion protein significantly inhibited the in vitro invasion of XGC9811-L cells, with the penetrating cells being significantly decreased compared with the control group per unit time (P?相似文献   

The cytotoxic effect of TGF-β1 on mesothelial cells via apoptosis in early peritoneal carcinomatosis

Peritoneal dissemination is one of the main causes of death in gastric cancer patients. We have previously reported that gastric cancer cells can induce peritoneal apoptosis, lead to damage of peritoneum integrity, and therefore promote peritoneal metastasis. However, the soluble factors secreted by cancer cells to trigger the damaging cascade remain unclear. TGF-β1, a cytokine known for its capacity to induce proliferative and transformative changes of cells is found in significantly higher quantities correlated with peritoneal metastasis and TNM stages of gastric cancer. High levels of TGF-β1 in the subperitoneal milieu may affect the morphology and function of mesothelial cells, so that the resulting environment becomes favorable for peritoneal metastases. We observed apoptosis induced by TGF-β1 in mesothelial cells in peritoneal carcinomatosis. Knockdown of the smad2 gene by siRNA silencing can partially inhibit these effects. TGF-β1 could upregulate the expressions of Bax and suppress Bcl-2 in mesothelial cells. We conclude that TGF-β1 could induce apoptosis of mesothelial cells, which involves the smad2 signaling pathway in peritoneal carcinomatosis. Bcl-2 and Bax may contribute to this phenomenon.     

Tumor-associated macrophages of the M2 phenotype contribute to progression in gastric cancer with peritoneal dissemination

Background

Tumor-associated macrophages (TAMs) of the M2 phenotype are known to promote tumor proliferation and to be associated with a poor prognosis in numerous cancers. Here, we investigated whether M2 macrophages participate in the development of peritoneal dissemination in gastric cancer.

Methods

The characteristics of peritoneal macrophages in gastric cancer patients with or without peritoneal dissemination were examined by flow cytometry and the real-time quantitative polymerase chain reaction. The effects of M2 macrophages on phenotypic changes of the gastric cancer cell line MKN45 were assessed with a direct or indirect co-culture system in vitro and an in vivo mouse xenograft model.

Results

The number of peritoneal macrophages with the M2 phenotype (CD68⁺CD163⁺ or CD68⁺CD204⁺) was significantly higher in gastric cancer patients with peritoneal dissemination than in those without peritoneal dissemination. Higher expression of the M2-related messenger RNAs (IL-10, vascular endothelial growth factor A, vascular endothelial growth factor C, matrix metalloproteinase 1, and amphiregulin) and lower expression of M1-related messenger RNAs (TNF-α, CD80, CD86, and IL-12p40) were also confirmed in the TAMs. Macrophage co-culture with gastric cancer cells converted M1 phenotype into M2 phenotype. Moreover, the coexistence of MKN45 cells with M2 macrophages resulted in cancer cell proliferation and an acceleration of tumor growth in the xenograft model.

Conclusions

Intraperitoneal TAMs in gastric cancer patients with peritoneal dissemination were polarized to the M2 phenotype, and could contribute to tumor proliferation and progression. Therefore, intraperitoneal TAMs are expected to be a promising target in the treatment of peritoneal dissemination in gastric cancer.

     

Transduction of soluble Flt-1 gene to peritoneal mesothelial cells can effectively suppress peritoneal metastasis of gastric cancer

The prognosis of gastric cancer with peritoneal metastasis has not improved. Despite many promising studies, gene therapy has limited clinical application because of the lack of suitable vector systems to enable selective gene transduction to tumor cells. The aim of this study was to clarify whether gene therapy targeted to peritoneal mesothelial cells (PMCs) can inhibit peritoneal dissemination of gastric cancer. In vitro experiments showed that adenovirus expressing LacZ infected human omental tissue-derived PMCs more efficiently than human gastric cancer cell lines MKN1 and MKN45. When adenovirus expressing LacZ was injected into the peritoneal cavity of nude mice, the expression was detected in the peritoneum for at least 4 weeks. Furthermore, when adenovirus expressing soluble Flt-1 (Ad-sFLT-1) was i.p. administered in vivo, a high level of sFlt-1 protein could be detected in peritoneal lavage for 8 weeks. When MKN45 cells were i.p. inoculated 3 days after adenoviral vector injection, Ad-sFLT-1 markedly reduced the number of metastatic nodules larger than 1 mm in diameter on the peritoneal surface, and significantly prolonged the survival of nude mice without any significant side effects. Thus, peritoneal dissemination was significantly suppressed by a single i.p. injection of Ad-sFlt-1. Anti-angiogenic gene therapy targeted to PMCs could be a novel and practical strategy against peritoneal dissemination of gastric cancer, because it does not require tumor-specific gene transfer.     

Peritoneal mesothelial cell injury factors in rat cancerous ascites

To elucidate the mechanism of the peritoneal dissemination of cancer, the influence of cancerous ascites on peritoneal mesothelial cells was studied by scanning electron microscopy and light microscopy. We inoculated normal Donryu rats with AH100B ascites hepatoma cells and studied the influence of the supernatant from cancerous ascites on the normal rat peritoneal surface by i.p. injection. The mesothelial cells were damaged and exfoliated markedly, which is supposed to be a profitable condition for cancer cells to proliferate on the peritoneal surface. Therefore, the presence of mesothelial cell injury factors was noted. Subsequently, we divided the supernatant from rat cancerous ascites into four fractions by gel filtration and revealed the distribution of mesothelial cell injury factors by studying the influence of each fraction on the normal rat peritoneal surface. Although Fraction I (fibrin fraction) and Fraction II (IgG fraction) made no changes on the peritoneal surface, Fraction III (albumin fraction) and Fraction IV provoked damages on the mesothelial cells. We found that the mesothelial cell injury factors are present in the albumin fraction and in the fraction containing low-molecular-weight substances.     

Dichloroacetate enhances apoptotic cell death via oxidative damage and attenuates lactate production in metformin-treated breast cancer cells

The unique metabolism of breast cancer cells provides interest in exploiting this phenomenon therapeutically. Metformin, a promising breast cancer therapeutic, targets complex I of the electron transport chain leading to an accumulation of reactive oxygen species (ROS) that eventually lead to cell death. Inhibition of complex I leads to lactate production, a metabolic byproduct already highly produced by reprogrammed cancer cells and associated with a poor prognosis. While metformin remains a promising cancer therapeutic, we sought a complementary agent to increase apoptotic promoting effects of metformin while attenuating lactate production possibly leading to greatly improved efficacy. Dichloroacetate (DCA) is a well-established drug used in the treatment of lactic acidosis which functions through inhibition of pyruvate dehydrogenase kinase (PDK) promoting mitochondrial metabolism. Our purpose was to examine the synergy and mechanisms by which these two drugs kill breast cancer cells. Cell lines were subjected to the indicated treatments and analyzed for cell death and various aspects of metabolism. Cell death and ROS production were analyzed using flow cytometry, Western blot analysis, and cell counting methods. Images of cells were taken with phase contrast microscopy or confocal microscopy. Metabolism of cells was analyzed using the Seahorse XF24 analyzer, lactate assays, and pH analysis. We show that when DCA and metformin are used in combination, synergistic induction of apoptosis of breast cancer cells occurs. Metformin-induced oxidative damage is enhanced by DCA through PDK1 inhibition which also diminishes metformin promoted lactate production. We demonstrate that DCA and metformin combine to synergistically induce caspase-dependent apoptosis involving oxidative damage with simultaneous attenuation of metformin promoted lactate production. Innovative combinations such as metformin and DCA show promise in expanding breast cancer therapies.