Human Renal Cell Carcinoma Cell Lines ACHN Research Articles

Pazopanib stimulates senescence of renal carcinoma cells through targeting nuclear factor E2-related factor 2 (Nrf2).

Renal cell carcinoma (RCC) is the most common kidney cancer with high mortality rate. Pazopanib has been approved for the treatment of RCC. However, the underlying mechanism is not clear. Here, we report a novel finding by showing that treatment with Pazopanib could promote cellular senescence of the human RCC cell line ACHN. Cells were stimulated with 5, 10, and 20 μM Pazopanib, respectively. Cellular senescence was measured using senescence-associated β-galactosidase (SA-β-Gal) staining. Western blot analysis and real-time polymerase chain reactionwere used to measure the mRNA and protein expression of nuclear factor E2-related factor 2 (Nrf2), γH2AX, human telomerase reverse transcriptase (hTERT), telomeric repeat binding factor 2 (TERF2), p53 and plasminogen activator inhibitor (PAI). First, we found that exposure to Pazopanib reduced the cell viability of ACHN cells. Additionally, Pazopanib induced oxidative stress by increasing the production of reactive oxygen species,reducing the levels of glutathione peroxidase,and promoting nuclear translocation of Nrf2. Interestingly, Pazopanib exposure resulted in DNA damage by increasing the expression of γH2AX. Importantly, Pazopanib increased cellular senescence and reduced telomerase activity. Pazopanib also reduced the gene expression of hTERT but increased the gene expression of TERF2. Correspondingly, we found that Pazopanib increased the expression of p53 and PAI at both the mRNA and protein levels. To elucidate the underlying mechanism, the expression of Nrf2 was knocked down by transduction with Ad- Nrf2 shRNA. Results indicate that silencing of Nrf2 in ACHN cells abolished the effects of Pazopanib in stimulating cellular senescence and reducing telomerase activity. Consistently, knockdown of Nrf2 restored the expression of p53 and PAI in ACHN cells. Based on these results, we explored a novel mechanism whereby which Pazopanib displays a cytotoxicity effect in RCC cells through promoting cellular senescence mediated by Nrf2.

Down-regulation of transient receptor potential melastatin member 7 prevents migration and invasion of renal cell carcinoma cells via inactivation of the Src and Akt pathway.

PurposeTransient receptor potential melastatin member 7 (TRPM7), an ion channel and serine/threonine protein kinase, has been linked with distinct human malignancies. However, the role of TRPM7 in renal cell carcinoma (RCC) has not been investigated. The aim of this study is to determine whether TRPM7 regulates the migration and invasion of RCC cells. Its relationship with signal transduction pathways was also studied.Materials and MethodsThe human RCC cell lines ACHN and SN12C were chosen for this study. The molecular mechanisms of TRPM7 action were studied using Western blot analysis and small interfering RNA (siRNA)-based knockdown. The effect of TRPM7 knockdown on RCC cells was measured by using Transwell invasion and wound healing migration assays.ResultssiRNA-induced silencing of TRPM7 notably decreased the migration and invasion of ACHN and SN12C RCC cells. The phosphorylation levels of Src in both cells were obviously reduced after TRPM7 silencing compared with that of the control ACHN and SN12C cells. Furthermore, the phosphorylation levels of Akt were greatly decreased in ACHN cells after siRNA-induced knockdown of TRPM7. Additionally, the treatment of cells with Src and Akt inhibitors clearly limited the migration and invasion of RCC cells.ConclusionsOur data show that TRPM7 regulated ACHN and SN12C RCC cell invasion via the Src/Akt signaling pathway. Therefore, targeting the Src/Akt signaling pathway and/or the expression or function of TRPM7 could be a potential beneficial treatment for patients with RCC.

BMP-2 inhibits tumor-initiating ability in human renal cancer stem cells and induces bone formation.

We have previously shown that BMP-2 induces bone formation and inhibits tumorigenicity of cancer stem cells (CSCs) in a human osteosarcoma OS99-1 cell line. In this study, we sought to determine whether BMP-2 can similarly induce bone formation and inhibit the tumorigenicity of renal CSCs identified based on aldehyde dehydrogenase (ALDH) activity in renal cell carcinoma (RCC) cell lines and primary tumors. Using a xenograft model in which cells from human RCC cell lines ACHN, Caki-2, and primary tumors were grown in NOD/SCID mice, renal CSCs were identified as a subset of ALDH(br) cells. The ALDH(br) cells possessed a greater colony-forming efficiency, higher proliferative output, increased expression of stem cell marker genes Oct3/4A, Nanog, renal embryonic marker Pax-2, and greater tumorigenicity compared to cells with low ALDH activity (ALDH(lo) cells), generating new tumors with as few as 25 cells in mice. In vitro, BMP-2 was found to inhibit the ALDH(br) cell growth, down-regulate the expression of embryonic stem cell markers, and up-regulate the transcription of osteogenic markers. In vivo, all animals receiving a low number of ALDH(br) cells (5 × 10(3)) from ACHN, Caki-2, and primary tumor xenografts treated with 30 µg BMP-2 per animal showed limited tumor growth with significant bone formation, while untreated cells developed large tumor masses without bone formation. These results suggest that BMP-2 inhibits the tumor-initiating ability of renal CSCs and induces osseous bone formation. BMP-2 may therefore provide a beneficial strategy for human RCC treatment by targeting the CSC-enriched population.

Abstract 2271: Effective therapy for advanced renal cell carcinoma using triptolide and TRAIL receptor agonists

Abstract Metastatic renal cell carcinoma (RCC) is incurable. IFNα or IL-2 immunotherapy has been used for RCC for more than 20 years, but response rates remain low and these cytokines can generate toxic. More recently, administration of multikinase inhibitors or anti-VEGF receptor mAb has become popular, but these agents are largely palliative and tumor regression is common. The shortcomings in current therapeutic options for patients with metastatic RCC provide the rationale for the development of novel treatment protocols. Triptolide, a diterpene triepoxide from the medicinal herb Tripterygium wilfordii, can suppress cell growth and induce apoptosis in a range of human tumor cells. Further, triptolide also sensitizes tumor cells to TNF-related apoptosis-inducing ligand (TRAIL). The objective of this study was to determine the effectiveness of combining triptolide with TRAIL receptor agonists against RCC. In vitro, the combination of triptolide and recombinant human TRAIL protein decreased cell viability in the human RCC cell line ACHN cells compared with either treatment alone. This decrease in viability correlated with increased induction of apoptosis (positive Annexin V staining) and caspase activation. The triptolide-induced sensitization was accompanied by decreased HSP70, HSP27, and HSF1 expression, as well as increased surface expression of TRAIL-R2. Similar in vitro treatment of the mouse renal cell adenocarcinoma cell line, Renca, demonstrated that triptolide enhanced the killing of these cells by membrane-bound TRAIL or agonistic anti-DR5 mAb. In vivo treatment of mice bearing orthotopic and metastatic Renca tumors showed that combination therapy consisting of minnelide (a water soluble version of triptolide) and agonistic anti-DR5 mAb resulted in significantly decreased tumor burden and increased animal survival compared to either therapy alone. Collectively, our data suggest that triptolide sensitizes RCC cells to TRAIL-induced apoptosis, which is accompanied by decreased heat shock protein expression and altered TRAIL receptor expression. These data also suggest that the combination of TRAIL receptor agonists and triptolide may prove to be an effective treatment for metastatic RCC, especially at doses that show suboptimal tumoricidal activity when used individually. Citation Format: Thomas S. Griffith, Erik L. Brincks, Tamara A. Kucaba, Britnie R. James, Veena Sangwan, Sulagna Banerjee, Ashok Saluja. Effective therapy for advanced renal cell carcinoma using triptolide and TRAIL receptor agonists. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2271. doi:10.1158/1538-7445.AM2014-2271

Development of a novel interferon-α2b gene construct with a repetitive hypoxia-inducible factor binding site and its suppressive effects on human renal cell carcinoma cell lines in vitro

Despite the advent of targeted therapies, interferon-alpha (IFN-α) remains a therapeutic option for advanced renal cell carcinoma (RCC), especially in Japan, with a treatment response rate of 15-20%. To improve the efficacy of IFN-α-based therapies, we evaluated a novel treatment strategy for RCC using an IFN-α2b gene construct with a repetitive hypoxia-inducible factor binding site. We constructed an expression plasmid designated 5HREp-IFN-α2b containing the coding region of the IFN-α2b gene. Five copies of the hypoxia-response element (HRE) sequences were inserted upstream of the IFN-α2b gene, and the construct was transfected into human RCC cell lines ACHN, 786-O and KU19-20. The concentrations of IFN-α2b in the conditioned media were measured by enzyme-linked immunosorbent assay. Cell viabilities were determined by MTS assays. Construct-induced IFN-α secretion was confirmed in all three cell lines. IFN-α production was significantly enhanced by the hypoxia-mimicking agent deferoxamine mesylate in cell lines expressing the wild-type von Hippel-Lindau (VHL) gene (KU19-20 and ACHN) compared with cells expressing the mutant VHL gene (786-O). The construct exerted significant suppressive effects on the viabilities of all RCC cell lines. This is the first study to report on the construction of a cytokine gene with a repetitive hypoxia-inducible factor binding site and its application in the suppression of human cancer cells. Gene therapy using this IFN-α2b gene construct with HREs may represent a novel treatment modality for advanced RCC.

A bone-seeking clone exhibits different biological properties from the ACHN parental human renal cell carcinoma in vivo and in vitro

Metastatic bone disease caused by renal cell carcinoma (RCC) occurs frequently. Very little is currently known about the mechanism of preferential metastasis of RCC to bone. We hypothesize that RCCs that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone-seeking (ACHN-BO) clones of the human RCC cell line ACHN by repeated four passages in nude mice and in vitro of metastatic cells obtained from bone. These clones were examined for distinguishing biological characteristics and compared with the ACHN parental cells (ACHN-P) in vivo and in vitro. Our results showed that the ACHN-BO cell line could be successfully obtained by in vivo selection through the lateral tail vein. This approach results in the development of multiple osteolytic lesions in the distal femora and proximal tibiae within four weeks after inoculation, with a success rate of 85–100% and no additional comorbidity. ACHN-P cells developed metastases in lung, bone, brain, ovary and adrenal glands. Conversely, ACHN-BO cells exclusively metastasized to bones with larger osteolytic lesions. Compared with the ACHN-P cell line, the proliferation ability in ACHN-BO6 was increased by 9.68 and 6.42%, respectively (P<0.05), while the apoptotic ratio decreased significantly (P<0.05) and cells were blocked in the S phase with suppressed migration and invasion capacities. The ACHN-BO6 cell line produced greater amounts of the pro-angiogenic factors VEGF and TGF-β than ACHN-P. Our data suggest that these phenotypic changes allow RCC cells to promote osteoclastic bone resorption, survive and proliferate in bone, which consequently leads to the establishment of bone metastases. This model provides a reliable reproduction of the clinical situation and, therefore, is suitable for designing and evaluating more effective treatments for RCC bone metastasis.

Abstract 4644: Significance of serum tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) levels of renal cell carcinoma patients as prognostic and monitoring biomarker

Abstract TRAIL and its receptors have been reported to play a significant role as cytotoxic and apoptotic mechanisms in cytotoxic T-lymphocytes and natural killer cells. TRAIL has been reported to induce apoptosis preferentially in various types of cancer cells, including renal cell carcinoma (RCC) cells. These findings suggest that TRAIL and its receptors might be useful biomarkers for RCC. In this study, we examined serum TRAIL levels of RCC patients and normal individuals and evaluated its utility as biomarker. Peripheral blood samples were obtained from 85 patients (60 men and 25 women, median age; 63 years [36-85]) with RCC before surgery. All the patients underwent radical or partial nephrectomy. The histological grade and TNM status distribution was as follows: Grade 1, n =1; Grade 2, n = 63 and Grade 3, n = 21; T1, n =56; T2, n = 8; T3, n =18 and T4, n = 3; N1-2, n = 7; and M1, n = 13. Blood samples were also collected from 52 healthy controls. The serum TRAIL levels were measured by a sandwich enzyme-linked immunosorbent assay (ELISA). The serum TRAIL levels in patients with RCC was lower than those of normal control individuals (79±8pg/ml vs 118±16 pg/ml, P =0.038). The serum TRAIL levels in RCC patients with lymph node metastasis (N1-2), distant metastasis (M1), microscopic venous invasion, and stage III-IV were significantly lower compared to those in RCC patients without lymph node metastasis (N0), without distant metastasis (M0), without microscopic venous invasion, and with stage I-II, respectively (P =0.051, P =0.045, P = 0.02, and P = 0.048). The serum TRAIL levels before and after surgery from the same patients (n = 17) was 61±15 pg/ml and 110±29 pg/ml, respectively, with significant difference (P = 0.015). The serum TRAIL levels after the surgery in these patients elevated to be similar to those detected in healthy controls (n = 52). The cause-specific survival rate of the RCC patients with high serum TRAIL was shown to be significantly higher than that of those with low serum TRAIL in the 5-year follow-up (P &amp;lt; 0.0385). The cytotoxicity of recombinant TRAIL and lymphocytes against human RCC cell line ACHN and primary culture RCC cell was measured by Crystal violet assay in the absence and presence of neutralizing anti-TRAIL antibody. TRAIL was estimated to contribute to about 20% of the lymphocyte-mediated cytotoxicity against ACHN and primary culture RCC cell. To our knowledge, the current study is the first to demonstrate the serum TRAIL levels of RCC patients in comparison with those of normal controls, in correlation with clinicopathological factors, and in association with postoperative prognosis. In conclusion, it is suggested that serum TRAIL levels in RCC patients might be utilized not only as diagnostic and prognostic biomarker but also as a possible biomarker for monitoring postoperative progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4644.

220 SIGNIFICANCE OF SERUM TUMOR NECROSIS FACTOR (TNF)-RELATED APOPTOSIS-INDUCING LIGAND (TRAIL) LEVELS AS PROGNOSTIC AND MONITORING BIOMARKER FOR RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research II1 Apr 2010220 SIGNIFICANCE OF SERUM TUMOR NECROSIS FACTOR (TNF)-RELATED APOPTOSIS-INDUCING LIGAND (TRAIL) LEVELS AS PROGNOSTIC AND MONITORING BIOMARKER FOR RENAL CELL CARCINOMA Daisuke Toiyama, Natsuki Takaha, Jun Fujiwara, Terukazu Nakamura, Humiya Hongo, Kazuya Mikami, Akihiro Kawauchi, and Tsuneharu Miki Daisuke ToiyamaDaisuke Toiyama More articles by this author , Natsuki TakahaNatsuki Takaha More articles by this author , Jun FujiwaraJun Fujiwara More articles by this author , Terukazu NakamuraTerukazu Nakamura More articles by this author , Humiya HongoHumiya Hongo More articles by this author , Kazuya MikamiKazuya Mikami More articles by this author , Akihiro KawauchiAkihiro Kawauchi More articles by this author , and Tsuneharu MikiTsuneharu Miki More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.277AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES TRAIL and its receptors have been reported to play a significant role as cytotoxic and apoptotic mechanisms in cytotoxic T-lymphocytes and natural killer cells. TRAIL has been reported to induce apoptosis preferentially in various cancer cells, including renal cell carcinoma (RCC) cells. These findings suggest that TRAIL and its receptors might be useful biomarkers for RCC. To our knowledge, no report on the serum TRAIL levels from RCC patients has been published. In this study, we examined serum TRAIL levels of RCC patients and normal individuals and evaluated its utility as biomarker. METHODS Peripheral blood samples were obtained from 85 patients (60 men and 25 women, median age; 63 years [36-85]) with RCC before surgery. All the patients underwent radical or partial nephrectomy. The histological grade and TNM status distribution was as follows: Grade 1, n =1; Grade 2, n = 63 and Grade 3, n = 21; T1, n =56; T2, n = 8; T3, n =18 and T4, n = 3; N1-2, n = 7; and M1, n = 13. Blood samples were also collected from 52 healthy controls. The serum TRAIL levels were measured by a sandwich enzyme-linked immunosorbent assay. The cytotoxicity of recombinant TRAIL and lymphocytes against human RCC cell line ACHN and primary culture RCC cell was measured by Crystal violet assay. RESULTS The serum TRAIL levels in patients with RCC was lower than those of normal control individuals (79 ± 9pg/ml vs 118 ± 17 pg/ml, P =0.02). The serum TRAIL levels in RCC patients with lymph node metastasis (N1-2), distant metastasis (M1), microscopic venous invasion, and stage II-IV were significantly lower compared to those in RCC patients without lymph node metastasis (N0), without distant metastasis (M0), without microscopic venous invasion, and with stage I, respectively (P <0.001, P =0.001, P = 0.008, and P = 0.004). The mean serum TRAIL levels before and after the surgery from the same patients (n = 17) was 61 ± 15 pg/ml and 110 ± 28 pg/ml, respectively (P = 0.015). The cause-specific survival rate of the RCC patients with high serum TRAIL was shown to be significantly higher than that of those with low serum TRAIL in the 5-year follow-up (P < 0.0385). TRAIL was estimated to contribute to about 20% of the lymphocyte-mediated cytotoxicity against ACHN and primary culture RCC cell. CONCLUSIONS These data suggest that serum TRAIL levels in RCC patients might be utilized not only as diagnostic and prognostic biomarker but also as a possible biomarker for monitoring postoperative progression. Kyoto, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e86 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daisuke Toiyama More articles by this author Natsuki Takaha More articles by this author Jun Fujiwara More articles by this author Terukazu Nakamura More articles by this author Humiya Hongo More articles by this author Kazuya Mikami More articles by this author Akihiro Kawauchi More articles by this author Tsuneharu Miki More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Enhanced susceptibility of adriamycin-treated human renal cell carcinoma cells to lysis by peripheral blood lymphocytes and tumor infiltrating lymphocytes

Previous studies indicated that the anticancer agent adriamycin (ADR) could induce activation of cytotoxic T lymphocytes (CTL) and natural killer cells. In this study, we investigated the effect of ADR on the susceptibility of human renal cell carcinoma (RCC) cells to lysis by peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). Treatment of human RCC cell line ACHN and freshly derived RCC cells with ADR at 1 microg/ml or more for 3 h significantly enhanced their susceptibility to lysis by PBL (P<0.05). This ADR-induced enhancement of susceptibility of RCC cells to lysis by PBL was also observed when freshly derived TIL were used as effector cells (P<0.05). ADR up-regulated the expression of leukocyte function-associated antigen-3 (LFA-3) and intercellular adhesion molecule-1 (ICAM-1), which are critical in the binding and killing of CTL against cancer cells. Of the five fresh RCC cell cultures treated with ADR, LFA-3 was increased in all and ICAM-1 was increased in three of them, respectively (P<0.05). Up-regulation of LFA-3 and ICAM-1 was also observed in ACHN cells treated with two derivatives of ADR, epirubicin and pirarubicin. ADR further significantly increased the bindings of PBL to RCC cells (P<0.05). These findings suggest that treatment of RCC patients with low doses of ADR may sensitize the RCC cells to killing by PBL and TIL and may be a novel immunotherapeutic modality for the treatment of drug-resistant and/or immune-resistant RCC. The inducing of LFA-3 and ICAM-1 by ADR may be involved in the enhancement of susceptibility of PBL and TIL-mediated cytolysis in human RCC cells.

Clinical significance of ST3Gal IV expression in human renal cell carcinoma

Alteration of sialyltransferase expression has been implicated in carcinogenesis. Out of sialyltransferases cloned to date, we focused on ST3Gal IV expression in human renal cell carcinoma (RCC). Levels of ST3Gal IV mRNA were examined in human RCC in comparison with non-tumor kidney. ST3Gal IV cDNA obtained by polymerase chain reaction from cDNA library of human RCC cell line ACHN was identical to STZ in nucleotide sequence. Northern blot analysis was performed for 24 non-tumor kidney and 25 primary RCC tissues, and 5 metastases. ST3Gal IV mRNA level was decreased in 16 cases of 22 primary RCC tissues compared to 21 non-tumor kidney tissues. The mRNA level was low in 4 and equivocal in one, of 5 metastases. The 6 cases that possessed almost the same levels of ST3Gal IV mRNA in primary tumor tissues as those in non-tumor kidneys showed favorable prognoses, as assessed by Kaplan-Meier curve. These results indicate that down-regulation of ST3Gal IV mRNA may be one of the factors associated with the malignant progression of human RCC.

Soluble Fas in serum from patients with renal cell carcinoma

Objectives. Fas/APO-1 is an apoptosis-signaling cell-surface receptor belonging to the tumor necrosis factor receptor family. The Fas-Fas ligand system plays an important role in cytotoxic T-lymphocyte-mediated or natural killer cell-mediated cytotoxicity against tumor cells. Soluble Fas (sFas), generated by alternative splicing, has been reported to antagonize the interaction of cell-surface Fas with Fas ligand. This study examined the level of sFas in the serum of patients with renal cell carcinoma (RCC) and investigated the correlation between the sFas level and clinicopathologic parameters of RCC. Methods. Using reverse transcriptase-polymerase chain reaction, we examined the production of sFas messenger RNA (mRNA) from the cultured human RCC cell lines ACHN and OUR-10 and from surgical specimens. We also measured sFas levels in the serum of 31 patients with RCC before and after nephrectomy using an sFas-specific enzyme-linked immunosorbent assay. Results. mRNA of sFas was identified both in cultured ACHN cells and human RCC tissues, although mRNA of wild-type Fas was exclusively predominant. The level of sFas in the serum of patients with RCC was significantly higher than that of normal controls, but sFas was not detectable in the supernatant of cultured renal cancer cells. Preoperative and postoperative serum sFas levels did not clearly correlate with the patients’ age or sex or with histologic stage, grade, or cell type of RCC. The serum sFas level in patients with RCC correlated with tumor size. In 24 of the 31 cases, radical nephrectomy reduced the serum sFas level within 3 months. Conclusions. Our results suggest that the elevated serum sFas level in patients with RCC might not be derived from the tumor itself but might reflect an immune response to the tumor burden. Serum sFas may be a useful indicator of tumor burden in patients with RCC.

Inverse relationship of expression between GM3 and globo-series ganglioside in human renal cell carcinoma.

Renal cell carcinoma (RCC) is highly metastatic. We previously showed that expression of globo-series ganglioside is associated with the metastatic potential of RCC. However, the mechanism of metastasis remains largely unknown, and there is no effective therapy for metastasis. It was recently shown that induction of differentiation of colon cancer cells by brefeldin A was accompanied by an increase of GM3 with a concomitant decrease of neolacto-series gangliosides. To get a clue to a new method of therapy for RCC, we investigated whether the similar changes occur in RCC cells expressing globo-series ganglioside. Growth suppression and an increase of GM3 simultaneous with a decrease of monosialosyl galactosyl globoside, a member of globo-series gangliosides, were observed in human RCC cell line ACHN following brefeldin A treatment. The resultant change of the ganglioside profile is inversely related to the ganglioside pattern associated with the malignant potential of RCC and almost coincided with that representative of RCC cases showing favorable prognoses. It is suggested that the inverse relationship of expression between GM3 and globo-series ganglioside is reflected on the degree of malignancy of RCC, and may be useful as one of the indicators for exploiting treatment methods of RCC.