Presence of CD44v9-Expressing Cancer Stem Cells in Circulating Tumor Cells and Effects of Carcinoembryonic Antigen Levels on the Prognosis of Colorectal Cancer.

Circulating tumor cells (CTCs) are cancer cells released from the primary tumor into the bloodstream, and contain cancer stem cells that influence tumor survival, recurrence, and metastasis. Here, we investigated CD44v9 expression in CTCs and impact of preoperative carcinoembryonic antigen (CEA) levels on colorectal cancer (CRC) prognosis. We analyzed the expression of CD44v9 mRNA in CTCs using reverse transcription-polymerase chain reaction and preoperative CEA levels in blood samples obtained from 300 patients with CRC. Subsequently, we evaluated the association of CD44v9 expression and CEA levels with clinicopathological factors. CD44v9 mRNA was expressed in 31.3% of the patients, and was significantly associated with liver metastasis. Patients with positive CD44v9 expression had a lower 5-year survival rate (62.3%) than those with negative CD44v9 expression (82.8%, p < 0.001). Cox regression analysis identified CD44v9 expression and high CEA levels (≥5 ng/mL) as poor prognostic factors, while negative CD44v9 expression and low CEA levels (<5 ng/mL) were associated with favorable prognosis (hazard ratio = 0.285, p = 0.006). These results suggest that a combination of CD44v9 mRNA expression in CTCs and serum CEA levels could serve as a valuable prognostic marker for CRC, potentially enhancing the accuracy of prognosis predictions.

Monitoring Metastatic Colorectal Cancer Progression According to Reactive Oxygen Metabolite Derivative Levels.

Oxidative stress has been implicated in the development, proliferation, and metastasis of colorectal cancer, but few studies have considered how oxidative stress changes in relation to treatment response. In this study, we investigated whether the rate of change in reactive oxygen metabolite derivatives (d-ROM)-serum markers of oxidative stress-could predict treatment response in metastatic colorectal cancer. We enrolled 53 patients with metastatic colorectal cancer who were treated with 3 months of chemotherapy. We measured d-ROM levels and performed computed tomography before and after chemotherapy, and we examined the change in d-ROM levels for each anticancer treatment. Factors influencing the d-ROM ratio (post-treatment: pre-treatment levels) were examined using linear regression analysis. d-ROM levels decreased in patients showing a partial response (p < 0.001) and increased in those showing disease progression (p = 0.042). An increasing d-ROM ratio was associated with disease progression (regression coefficient: 0.416, 95% confidence interval: 0.279-0.555, p < 0.001). Our study indicates that d-ROM levels are useful markers of tumor progression and that the d-ROM ratio is useful for predicting treatment response in patients with metastatic colorectal cancer.

Hepatic artery infusion therapy is effective for chemotherapy-resistant liver metastatic colorectal cancer.

BACKGROUND: Systemic FOLFOX (folinic acid (leucovorin (LV)), 5-fluorouracil (5-FU), and oxaliplatin), FOLFIRI (LV, 5-FU, and irinotecan), or FOLFOXIRI (5-FU, leucovorin, oxaliplatin, and irinotecan) chemotherapy regimens and additional molecular-target treatments, including anti-vascular endothelial growth factor, anti-epidermal growth factor receptor, and anti-multi-kinase antibodies, have been recommended for unresectable recurrent colorectal cancers. However, no effective treatments are currently available for cases refractory to these therapies. Therefore, the development of alternative therapies is desired. In the present study, we administered and observed the effectiveness of hepatic artery infusion therapy (HAIC) in patients with unresectable liver metastatic colorectal cancers refractory to systemic chemotherapy. In addition, we observed that in an experimental system with anticancer drug-resistant colorectal cancer lines, apoptosis and cell death could be induced by increasing anticancer drug concentrations. METHODS: The subjects had liver metastatic colorectal cancers that were unresponsive to systemic chemotherapy (FOLFOX/FOLFIRI) or to additional molecular-target therapies for progressive disease. Hepatic infusion tube placement was conducted according to the Seldinger method to insert a catheter with a side hole via the right femoral artery. A coiling procedure was performed to prevent drug influx into the gastroduodenal artery. Ten subjects were selected, and the results were evaluated after HAIC (5-FU and LV administered once weekly). Moreover, anticancer drug-resistant colorectal cancer lines were subsequently prepared to investigate whether increased anticancer drug concentrations could induce apoptosis or cell death. RESULTS: Of the 10 subjects, 3 (30 %) showed partial response and 4 (40 %) showed no change according to computed tomography imaging findings obtained after hepatic artery infusion. The disease control rate was 70 %. Eight subjects had improved quality of life. Survival time ranged from 2 to 16 months (median, 9 months). Meanwhile, we found that higher anticancer drug concentrations induced apoptosis and cell death in an anticancer drug-resistant colorectal cancer cell line. CONCLUSIONS: HAIC was effective in some systemic chemotherapy-resistant colorectal cancers with liver metastases and should be considered as an effective palliative therapy. This supports the finding that apoptosis and cell death could be induced in anticancer drug-resistant colorectal cancer cells in a drug concentration-dependent manner.

Prokineticin 2 (PROK2) is an important factor for angiogenesis in colorectal cancer.

The Prokineticin 2 (PROK2) is correlated with indispensable in maintaining the homeostasis of healthy human tissues. Herein, we examined the role of PROK2 in human colorectal cancer.After total RNA extraction from 6 colorectal cancer cell lines, we examined the expression of PROK2 mRNA. For investigating angiogenesis and tumor growth in mice, the PROK2 gene was transfected into colorectal cancer cell lines having low PROK2 mRNA expression. In addition, small interfering RNA (siRNA) was transfected into colorectal cancer cell lines having high PROK2 mRNA expression for investigation of angiogenesis and tumor growth in mice.From 6 colorectal cancer cell lines studied, PROK2 mRNA expression was increased in 3 cell lines. When the PROK2 gene was transfected into the colorectal cancer cell line with low PROK2 mRNA expression, angiogenesis and tumor growth in mice increased significantly compared to the cell line with the control vector.When PROK2 siRNA was transfected into colorectal cancer cell lines with high PROK2 mRNA expression, angiogenesis and tumor growth in mice were suppressed significantly compared to the cell line with siRNA (control).This is the first report of the association of PROK2 as an angiogenic growth factor in colorectal cancer.

Cancer stem cell marker in circulating tumor cells: expression of CD44 variant exon 9 is strongly correlated to treatment refractoriness, recurrence and prognosis of human colorectal cancer.

BACKGROUND/AIM: The expression of the CD44 variant exon 9 (CD44v9) was investigated in order to elucidate its significance for cancer stem cells in circulating human colorectal cancer cells (CTCs). MATERIALS AND METHODS: After peripheral blood was drawn from patients with colorectal cancer, CTCs were collected. Using the reverse transcription-polymerase chain reaction method, we examined the relationship between expression of CD44v9 mRNA and prognosis. RESULTS: In 60 out of 150 patients with colorectal cancer, expression of CD44v9 mRNA was positive in CTCs. In patients with stage III disease, the 5-year survival rate was 89% for patients with negative CD44v9 expression, whereas it was 52.4% in patients with positive expression (p<0.05). In patients with stage IV unresectable cancer, the 2-year survival rate was 70.1% in cases with CD44v9-negative expression and 33.3% in cases of positive expression (p<0.05). CONCLUSION: CD44v9 mRNA in the CTCs of colorectal cancer is useful as a factor predicting recurrence, prognosis, and treatment efficacy.

CD44variant exon 9 plays an important role in colon cancer initiating cells.

Cancer stem cells(cancer initiating cells) have become increasingly important in the treatment of malignant tumors. CD44 in particular has been identified as a marker for stem cells in colon cancer, which is a high-morbidity tumor. However, many details remain unknown, including identification of the relevant exon. The elucidation of these details could lead to the development of new therapies and improvements in prognosis. We report our findings on the importance of CD44 variant exon 9(v9) of stem cells in colon cancer. Using the anti-CD44 standard form(s) antibody, as well as antibodies for each of the CD44 variant exons, we studied colon cancer cell lines by examining stained images of stem cells in the crypt of normal colon mucosa. Using the anti-CD44v9 antibody that fits the normal colon mucosa stem cells, we screened cells using flow cytometry to examine colony formation, resistance to anticancer drugs, and tumor mass formation after subcutaneous implantation in mice. The stem cell-containing region in the crypt of normal colon mucosa was negative for anti-Ki67 antibody staining; only the anti-CD44 v9 antibody stain was expressed. As for colony formation, resistance to anticancer drugs, and tumor mass formation, cells positive both for anti-CD44s and anti-CD44v9 antibody stains was significantly more frequent than those positive for anti-CD44s antibody stain and negative for anti-CD44v9 antibody stain and those negative both for anti-CD44s and anti-CD44v9 antibody stains. CD44 variant exon 9 plays an important role in colon cancer stem cells.

Changes in CO2 concentration increase the invasive ability of colon cancer cells.

BACKGROUND/AIM: We studied the effects of CO(2) concentration changes on the invasive ability of colon cancer cells. MATERIALS AND METHODS: Colon cancer cell lines and human samples derived from a peritoneal metastasis were incubated in a hypercapnic environment, followed by incubation in 5% CO(2). The invasive ability of colon cancer cells incubated with CO(2) were analyzed using an invasion assay system. RESULTS: In comparison with the colon cancer cell lines incubated in 5% CO(2) only, the invasive ability of cells increased in all the colon cancer cell lines subjected to incubation in 20% CO(2) followed by incubation in 5% CO(2), with a concomitant increase in the mRNA expressions of matrix metalloproteinase-2 (MMP2) and MMP9. The invasive capability of peritoneal metastatic cells in the human-derived specimen also increased on CO(2) concentration changes. CONCLUSION: CO(2) concentration changes enhanced the invasive capacity of colon cancer cells.

The expression of integrins is decreased in colon cancer cells treated with polysaccharide K.

Polysaccharide K (PSK), a protein-bound polysaccharide used as a non-specific immunotherapeutic agent, is said to improve the prognosis of malignant tumors such as colon cancer, but there have been few in­depth investigations of its mechanism of action. In the present study, we investigated which genes in colon cancer cells themselves are regulated by PSK and what sort of action it exerts. Colon cancer cells were exposed to PSK in vitro and changes to their morphology and adhesive capacity were investigated, as were the changes in integrin expression that occurred. Exposure to PSK caused colon cancer cells to become spherical in morphology and investigation of their adhesion rate showed that it reduced adhesion to laminin, fibrinogen, collagen IV, collagen I and fibronectin. In terms of changes to molecules on the surface of cancer cells, there was reduced expression of integrin mRNA α and ß, which are ligands for the proteins of basement membrane. An investigation of the adhesiveness of PSK-stimulated colon cancer cells to vascular endothelial cells also showed that the adhesion rate decreased significantly compared with cells not exposed to PSK. Suppression of integrin expression on the cell surface and reduced adhesion to vascular endothelial cells were observed as a novel mechanism of action of PSK on colon cancer cells.

Endocrine gland-derived vascular endothelial growth factor strengthens cell invasion ability via prokineticin receptor 2 in colon cancer cell lines.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) has recently been identified as one of the vascular endothelial growth factors, and it is considered that the overexpression of EG-VEGF in colon cancer is related to hepatic metastasis. In this study, we report our recent novel findings of the involvement of EG-VEGF in cell invasion of colon cancer cells. Colon cancer cell lines (DLD-1 and HCT116) with high expression of prokineticin receptor (PK-R) 1 and 2 were stimulated with the EG-VEGF protein. Furthermore, Matrigel cell invasion assay was performed to examine the changes in cancer cell invasion. In addition, we investigated the mRNA expression of matrix metalloproteinase (MMP)-2, -7 and -9 in cancer cells. Finally, the EG-VEGF receptor on the colon cancer cell membrane was blocked by anti-PK-R1 and -PK-R2 antibodies to study whether cell invasion ability would be altered. In colon cancer cell lines where the expression of PK-R1 and 2 was confirmed, stimulation with EG-VEGF increased cell invasion a maximum of ~3-5 times. Furthermore, an increase in the mRNA and protein expression of MMP-2, -7 and -9 was observed. We also observed that the cell invasion rate decreased only after exposure to the anti-PK-R2 antibody. The study showed that the EG-VEGF protein may act on MMP-2, -7 and -9 via PK-R2 to strengthen cell invasion ability in colon cancer cell lines.

Polysaccharide K suppresses angiogenesis in colon cancer cells.

The protein-bound polysaccharide K (PSK) is used as a non-specific immunotherapeutic agent for the treatment of colon cancer. Little research, however, has been conducted on its association with angiogenesis, which is a prognostic factor markedly correlated with hematogenous metastases. We therefore decided to investigate the action of PSK on angiogenic growth factors, angiogenesis inhibitors and angiogenesis in colon cancer cells. Reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate changes in HIF-1α mRNA expression. PCR array was used to investigate changes in angiogenic growth factors and angiogenesis inhibitors, as well as the expression of related genes. Colon cancer cells were cultured with or without PSK for 48 h. The following day, cells were cultured for two days at 37°C in new complete media. The resulting culture medium was placed in the chamber of a tube formation system in order to investigate tube formation. Investigation of HIF-1α mRNA expression in colon cancer cell lines and in cells cultured under identical conditions with added PSK revealed a significant decrease in expression, as well as a decrease in angiogenic growth factors and related genes in PSK-treated colon cancer cell lines. By contrast, levels of angiogenesis inhibitors and related genes were higher in the PSK-treated colon cancer cell lines. Investigation of tube formation revealed that elongation was inhibited in the medium of the PSK-treated colon cancer cell lines in comparison to the medium of the non-treated colon cancer cell lines. PSK suppresses angiogenic growth factors and related genes, enhances angiogenesis inhibitors and related genes and ultimately suppresses angiogenesis in colon cancer cells.

[A case of response to panitumumab as third-line chemotherapy for multiple liver metastases and portal venal tumor embolus of rectal cancer].

A 64-year-old man who underwent rectal amputation for rectal cancer was diagnosed with multiple liver metastases and tumor embolus in the portal vein 6 months after operation. Though the patient underwent chemotherapy, mFOLFOX6, and bevacizumab+FOLFIRI, liver metastases were diagnosed as progressive disease (PD). After panitumumab+FOLFIRI was administered for three months as third-line chemotherapy, the tumor embolus completely disappeared, and liver metastases became cytoreductive on CT. The patient was judged to have achieved a partial response (PR). This case indicated that panitumumab was effective as third-line chemotherapy for unresectable recurrent rectal cancer.

MUC2 protein expression status is useful in assessing the effects of hyperthermic intraperitoneal chemotherapy for peritoneal dissemination of colon cancer.

We conducted a molecular biological investigation to determine the outcomes of hyperthermic intraperitoneal chemotherapy (HIPEC) treatment, and whether it is effective in all cases for patients with peritoneal dissemination of colon cancer. In the HIPEC group, the 3-year survival rate was 39.2%, whereas in the non-HIPEC group the 3-year survival rate was 15.6%. MUC2 expression was investigated in the HIPEC group, in patients positive for MUC2 expression, and the 3-year survival rate was 0.0%, while in patients negative for MUC2 expression, the 3-year survival rate was 61.1%. In addition, as a result of introducing MUC2-siRNA into a colon cancer cell line with high expression of the MUC2 gene, the cell death rate from heat and anticancer agents increased 40% in comparison with colon cancer cells in which scrambled siRNA had not been introduced. HIPEC therapy is thought to be effective in prolonging survival in patients with peritoneal dissemination of colon cancer, and MUC2 expression is thought to be useful as an indicator to assess its effectiveness in colon cancer cells.