VEGF 165 b, an antiangiogenic VEGF-A isoform, binds and inhibits bevacizumab treatment in experimental colorectal carcinoma: balance of pro- and antiangiogenic VEGF-A isoforms has implications for therapy

Bevacizumab, an anti-vascular endothelial growth factor (VEGF-A) antibody, is used in metastatic colorectal carcinoma (CRC) treatment, but responses are unpredictable. Vascular endothelial growth factor is alternatively spliced to form proangiogenic VEGF(165) and antiangiogenic VEGF(165)b. Using isoform-specific enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, we found that over 90% of the VEGF in normal colonic tissue was VEGF(xxx)b, but there was a variable upregulation of VEGF(xxx) and downregulation of VEGF(xxx)b in paired human CRC samples. Furthermore, cultured colonic adenoma cells expressed predominantly VEGF(xxx)b, whereas colonic carcinoma cells expressed predominantly VEGF(xxx). However, adenoma cells exposed to hypoxia switched their expression from predominantly VEGF(xxx)b to predominantly VEGF(xxx). VEGF(165)b overexpression in LS174t colon cancer cells inhibited colon carcinoma growth in mouse xenograft models. Western blotting and surface plasmon resonance showed that VEGF(165)b bound to bevacizumab with similar affinity as VEGF(165). However, although bevacizumab effectively inhibited the rapid growth of colon carcinomas expressing VEGF(165), it did not affect the slower growth of tumours from colonic carcinoma cells expressing VEGF(165)b. Both bevacizumab and anti-VEGF(165)b-specific antibodies were cytotoxic to colonic epithelial cells, but less so to colonic carcinoma cells. These results show that the balance of antiangiogenic to proangiogenic isoforms switches to a variable extent in CRC, regulates tumour growth rates and affects the sensitivity of tumours to bevacizumab by competitive binding. Together with the identification of an autocrine cytoprotective role for VEGF(165)b in colonic epithelial cells, these results indicate that bevacizumab treatment of human CRC may depend upon this balance of VEGF isoforms.     

Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells

Vascular endothelial growth factor (VEGF) is associated with tumor angiogenesis and poor prognosis in human colorectal cancer (CRC). VEGF receptor-1 (VEGFR-1 or Flt-1) is a high-affinity receptor for VEGF and is typically considered specific to endothelial cells. Here we report the expression and function of VEGFR-1 in CRC cell lines. VEGFR-1 was expressed in all CRC cell lines studied as determined by RT-PCR, Western blot analysis, FACS, and ELISA. Treatment of the human CRC cell lines HT-29 and SW480 with VEGF-A (a ligand for both VEGFR-1 and -2) or VEGF-B (a ligand specific for VEGFR-1) led to activation of Erk-1/2, SAPK/JNK, and translocation of the p65 subunit of nuclear factor-kappaB into the nucleus. Both VEGF-A and -B led to significant induction of cell motility and invasiveness of CRC cells. Stimulation of cells with VEGF-A or -B also led to larger and more numerous colonies in soft agar. However, activation of VEGFR-1 did not increase CRC cell proliferation. In contrast to the previous paradigm that VEGFRs are not present on tumor cells of epithelial origin, we found that VEGFR-1 is present and functional on CRC cells, and activation by VEGF family ligands can activate processes involved in tumor progression and metastasis.     

All-trans retinoic acid induces cellular senescence via upregulation of p16, p21, and p27

We here present a new anti-tumor mechanism of all-trans retinoic acid (ATRA). ATRA induced several biomarkers of cellular senescence including irreversible G1 arrest, morphological changes, senescence-associated β-galactosidase, and heterochromatin foci in HepG2 cells. ATRA also upregulated levels of p16, p21, and p27 which lead to activation of Rb and subsequent inactivation of E2F1. These effects were abolished by the RNA interference-mediated silencing of p16, p21, and p27. Moreover, ATRA failed to induce cellular senescence in Huh7 and HCT116, in which p16, p21, and p27 were not upregulated by ATRA, confirming that ATRA induces cellular senescence via upregulation of p16, p21, and p27.     

Intrinsic bevacizumab resistance is associated with prolonged activation of autocrine VEGF signaling and hypoxia tolerance in colorectal cancer cells and can be overcome by nintedanib,a small molecule angiokinase inhibitor

Colorectal cancer (CRC) is a common tumor type with a high mortality rate, in part due to intrinsic drug resistance. Although bevacizumab, a VEGF-directed neutralizing antibody, is particularly active in this pathology, some patients never respond for reasons not well understood. We here wish to clarify the role of autocrine VEGF signaling in the response of CRC cells to angiogenesis inhibition. Our results show that CRC cells with intrinsic bevacizumab-resistance displayed pronounced upregulation of autocrine HIF-VEGF-VEGFR signaling in response to prolonged bevacizumab exposure whereas the same signaling pathway was downregulated in bevacizumab-sensitive xenografts. Importantly, both bevacizumab-sensitive and -resistant CRC xenografts were sensitive to nintedanib, a small molecule angiokinase inhibitor, which was associated with inhibition of mTORC1. In vitro studies revealed that bevacizumab-resistant cells displayed intrinsically higher HIF-VEGF signaling intensity and hypoxia tolerance compared to their bevacizumab-sensitive counterparts. Interestingly, although nintedanib showed comparable activity toward bevacizumab-sensitive cells under normoxia and hypoxia, the drug was three-fold more toxic to the resistant cells under hypoxia, suggesting that nintedanib attenuated the survival signaling that usually protects these cells from hypoxia-mediated cell death. In conclusion, our findings support a role for autocrine VEGF signaling in the survival of CRC cells to hypoxia and thus to angiogenesis inhibition. We further show that nintedanib, a small molecule angiokinase inhibitor, is active toward CRC models with intrinsic bevacizumab resistance supporting clinical trials of nintedanib in patients that do not respond to bevacizumab, alone or in combination with bevacizumab to increase angiogenesis inhibition.     

Colorectal carcinoma cell production of transforming growth factor beta decreases expression of endothelial cell vascular endothelial growth factor receptor 2

BACKGROUND:

Vascular endothelial growth factor (VEGF) signaling is a target for antiangiogenic cancer therapy. The authors have previously observed that up to 40% of vessels in colorectal carcinoma (CRC) tumors are negative for VEGF receptor 2 (VEGFR2) expression. Differential activity of transforming growth factor beta (TGF‐β) is a potential contributor to this receptor heterogeneity because TGF‐β contributes to both angiogenesis and CRC tumor progression.

METHODS:

The authors analyzed VEGFR2 expression by Western blotting, and TGF‐β expression in endothelial and CRC cell lines, respectively. In addition, they immunostained endothelial cells in CRC xenografts to find an association between VEGFR2 and TGF‐β levels or activity.

RESULTS:

In bovine aortic endothelial cells (BAECs), TGF‐β1 significantly repressed VEGFR2 protein in a time‐dependent and dose‐dependent fashion (P < .05). Serum‐free conditioned media from various malignant human CRC cell lines (HCT116, 379.2, Dks8, and DLD1) induced down‐regulation of VEGFR2 in BAECs. This effect was proportional to the total levels of TGF‐β1 and TGF‐β2 and was blocked by SB‐431542 and SD‐208, TGF‐β receptor I inhibitors. Immunofluorescence staining of subcutaneous mouse xenografts of HCT116, 379.2, Dks8, and SW480 cells revealed vessels with an inverse relationship between TGF‐β activity and VEGFR2 expression. Oxygen and bone morphogenetic protein 9 levels were shown to modulate TGF‐β–induced VEGFR2 down‐regulation.

CONCLUSIONS:

In combination with other factors, TGF‐β may contribute to the vascular heterogeneity in human colorectal tumors. Cancer 2011;. © 2011 American Cancer Society.     

Functions and Clinical Implications of Autocrine VEGF Signaling in Colorectal Cancer

Vascular endothelial growth factor (VEGF) signaling is linked to invasiveness and aggressive disease in colorectal cancer (CRC), and two VEGF-directed agents, bevacizumab and aflibercept, are currently approved for treatment of metastatic disease. CRC cells are a major source of VEGF that is secreted into the tumor environment where it can associate with VEGF receptors on neighboring endothelial cells thereby promoting tumor angiogenesis. In addition, the CRC cells express functional VEGF receptors giving rise to autocrine VEGF signaling. So far, the biological and clinical implications of autocrine VEGF signaling have attracted less attention than the classical paracrine VEGF signaling pathways. In this review we focus on the effect of autocrine VEGF signaling on the survival, invasion, and drug-resistance of CRC cells and on the prognostic value of expression of VEGF ligands and receptors in CRC patients. We then discuss the different factors associated with a therapeutic response to VEGF blockage and summarize future challenges.     

Effect of VEGF receptor inhibitor PTK787/ZK222584 [correction of ZK222548] combined with ionizing radiation on endothelial cells and tumour growth.

The vascular endothelial growth factor (VEGF) receptor is a major target for anti-angiogenesis-based cancer treatment. Here we report the treatment effect of ionizing radiation in combination with the novel orally bioavailable VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on endothelial cell proliferation in vitro and with tumour xenografts in vivo. Combined treatment of human umbilical vein endothelial cells with increasing doses of PTK787/ZK222584 and ionizing radiation abrogated VEGF-dependent proliferation in a dose-dependent way, but inhibition of endothelial cell proliferation was not due to apoptosis induction. In vivo, a combined treatment regimen of PTK787/ZK222584 (4 x 100 mg/kg) during 4 consecutive days in combination with ionizing radiation (4 x 3 Gy) exerted a substantial tumour growth delay for radiation-resistant p53-dysfunctional tumour xenografts derived from SW480 colon adenocarcinoma cells while each treatment modality alone had only a minimal effect on tumour size and neovascularization. SW480 tumours from animals that received a combined treatment regimen, displayed not only an extended tumour growth delay but also a significant decrease in the number of microvessels in the tumour xenograft. These results support the model of a cooperative anti-tumoral effect of angiogenesis inhibitor and irradiation and show that the orally bioavailable VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 is suitable for combination therapy with irradiation.     

Genetic cooperation between p21Cip1 and INK4 inhibitors in cellular senescence and tumor suppression

Cell-cycle inhibitors of the Cip/Kip and INK4 families are involved in cellular senescence and tumor suppression. Some of these proteins, p21(Cip1), p16(INK4a) and p15(INK4b), are coexpressed in response to antiproliferative signals such as cellular senescence resulting in cell-cycle arrest. To understand the roles of these inhibitors and their synergistic effect, we have characterized the growth properties and senescent behavior of primary cells deficient in p21(Cip1) and expressing an endogenous Cdk4(R24C) (cyclin-dependent kinase) mutant (Cdk4(R24C) knock-in cells) insensitive to INK4 proteins. Inactivation of both p21(Cip1) and INK4 pathways strongly cooperate in suppressing cellular senescence in vitro. These double mutant cells behavior as immortal cultures and display high sensitivity to cellular transformation by oncogenes. Moreover, mice double mutant in the INK4 and p21(Cip1) pathways (Cdk4(R24C); p21(Cip1)-null mice) display an increased incidence of specific sarcomas, suggesting a significant cooperation between these two families of cell-cycle inhibitors in senescence responses and tumor suppression in vivo.     

Senescence and telomere shortening induced by novel potent G-quadruplex interactive agents, quindoline derivatives, in human cancer cell lines

Agents stabilizing G-quadruplexes have the potential to interfere with telomere replication by blocking the elongation step catalysed by telomerase or telomerase-independent mechanism and could therefore act as antitumor agents. In this study, we found that quindoline derivatives interacted preferentially with intramolecular G-quadruplex structures and were novel potent telomerase inhibitors. Treatment with quindoline derivatives reproducibly inhibited telomerase activity in human leukemia K562 cells and colon cancer SW620 cells. N'-(10H-Indolo [3,2-b] quinolin-11-yl)-N, N-dimethyl-propane-1,3-diamine (SYUIQ-5), (one of quindoline derivatives), when added to K562 and SW620 cell culture at nonacute cytotoxic concentrations, increased time of population doublings of K562 and SW620 cells, induced a marked cessation in cell growth and cellular senescence phenotype after 35 and 18 days, respectively. Growth cessation was accompanied by a shortening of telomere length, and induction of p16, p21 and p27 protein expression. However, another compound SYUIQ-7 with greater IC(50) for telomerase had no obvious cellular effect in nonacute cytotoxic concentrations. These results indicate that quindoline derivatives as novel potent G-quadruplex interactive agents induce senescence and telomere shortening in cancer cells and therefore are promising agents for cancer treatment.     

Oxidative stress‐dependent increase in ICAM‐1 expression promotes adhesion of colorectal and pancreatic cancers to the senescent peritoneal mesothelium

Intercellular adhesion molecule‐1 (ICAM‐1) has been implicated in adhesion of colorectal and pancreatic cancer cells (of the SW480 and PSN‐1 line, respectively) to the peritoneal mesothelium. It has been demonstrated that ICAM‐1 expression increases with senescence in some cell types, however, the significance of this phenomenon in the context of malignant dissemination remains elusive. In this report we show that the adherence of SW480 and PSN‐1 cells to senescent human omentum‐derived mesothelial cells (HOMCs) in vitro is greater than to early‐passage cells and that the effect is mediated by ICAM‐1. Senescent HOMCs display increased expression of ICAM‐1 mRNA and cell surface protein. The development of this phenotype is related to increased oxidative stress in senescent cells. The augmented ICAM‐1 expression in HOMCs can be reduced by culturing cells with antioxidants; in contrast, exposure of HOMCs to an oxidant, t‐BHP, leads to cellular senescence and increased ICAM‐1 expression. The effect is partly mediated by activation of p38 MAPK and AP‐1 signaling pathways. Finally, culture of HOMCs in the presence of a strong antioxidant, PBN, significantly reduces the senescence‐associated increase in SW480 and PSN‐1 cancer cell binding. These results indicate that increased oxidative stress and increased expression of ICAM‐1 in senescent HOMCs may facilitate peritoneal adhesion of selected colorectal and pancreatic cancers.     

Evidence that senescent human prostate epithelial cells enhance tumorigenicity: cell fusion as a potential mechanism and inhibition by p16INK4a and hTERT

Normal human prostate (NHP) epithelial cells undergo senescence in vitro and in vivo but the potential role of senescent NHP cells in prostate tumorigenesis remain unclear. Here we show that senescent NHP cells enhance the in vivo tumorigenicity of low-tumorigenic LNCaP prostate cancer and low/non-tumorigenic subset of cells (called L cells) isolated from multiple bulk-cultured prostate (and other) cancer cell lines. Subsequent studies suggest cell-cell fusion as a potential mechanism for senescent NHP cell-enhanced tumor development. Using fluorescently tagged tumor cells and/or NHP cells, we find that NHP cells, like fibroblasts, can undergo fusion with unfractionated tumor cells or the L cells. Using 293T-L cells as the model cell system, we verify NHP and 293T-L cell fusion by using differential RT-PCR, karyotyping, and gene expression analyses. Further experiments demonstrate that senescent NHP cells that have lost progenitor markers, accumulated p16INK4a (p16) protein expression, and acquired the AR mRNA expression, appear to be the preferential fusion targets. Strikingly, the tumorigenicity of the NHP/293T-L hybrid cells was inhibited by exogenous p16 as well as hTERT. Chromosomal analyses revealed that hTERT probably inhibited the in vivo tumorigenicity by maintaining genomic stability. These results suggest that senescent NHP cells, like senescent fibroblasts, may promote tumor development and that one of the mechanisms underlying the senescent NHP cell-enhanced tumorigenicity could be through cell fusion.     

The vascular endothelial growth factor receptor (VEGFR-1) supports growth and survival of human breast carcinoma

Vascular endothelial growth factor receptor 1 (VEGFR-1) is present on endothelial cells and subsets of human tumor cells, raising the hypothesis that angiogenic factors may promote tumor growth both by inducing angiogenesis and directly signaling through activation of VEGFR-1 on tumor cells. Here, we report that VEGFR-1 is expressed on a panel of 16 human breast tumor cell lines, and the vasculature and the tumor cell compartment of a subset of breast carcinoma lesions, and that selective signaling through VEGFR-1 on breast cancer cells supports tumor growth through downstream activation of the p44/42 mitogen-activated protein kinase (MAPK) or Akt pathways. Ligand-stimulated proliferation of breast tumor cells was inhibited by specific blockade with an anti-VEGFR-1 neutralizing monoclonal antibody. Treatment with anti-VEGFR-1 mAb significantly suppressed the growth of DU4475, MCF-7, BT-474 and MDA-MB-231 breast xenografts in athymic mice. Histological examination of anti-VEGFR-1 mAb treated tumor xenografts showed a significant reduction of activation of the p44/42 MAPK or Akt pathways in tumor cells resulting in an increase in tumor cell apoptosis. Importantly, cotreatment with mAbs targeting human VEGFR-1 on tumor cells and murine VEGFR-1 on vasculature led to more potent growth inhibition of breast tumor xenografts. The results suggest that VEGF receptors may not only modulate angiogenesis, but also directly influence the growth of VEGF receptor expressing tumors.     

Abrogation of VEGF expression in human head and neck squamous cell carcinoma decreases angiogenic activity in vitro and in vivo

Angiogenesis is increased in various human cancers, including head and neck squamous cell carcinoma (HNSCC), and correlates with tumor progression and metastasis. Vascular endothelial growth factor (VEGF) has been shown to be a key regulator of angiogenesis. We determined whether VEGF antisense oligonucleotide treatment can decrease angiogenic activity of HNSCC cell lines in vitro and of HNSCC xenografts in vivo. Established human HNSCC cell lines were screened for VEGF expression at both mRNA and protein levels. By using a 21-mer antisense phosphorothioate oligonucleotide targeting the translation start site of human VEGF mRNA, we examined modulation of VEGF expression in cell line supernatants by capture ELISA, and in cell lysates by Western blotting. Human umbilica vein endothelial cells (HUVEC) were grown in conditioned medium produced from the treated tumor cells. Endothelial cell (EC) proliferation was determined by cell count and EC migration was measured using a modified Boyden chamber. Mice with HNSCC xenografts were treated with PBS, VEGF antisense or sense oligonucleotides (10 mg/kg; i.p. injection), respectively and tumor volumes were measured for 5 weeks. VEGF antisense oligonucleotide treatment resulted in a significant reduction of VEGF protein expression compared to sense control. Although the growth rate of the tumor cell lines was not affected, addition of conditioned medium from VEGF antisense-treated tumor cells resulted in decrease of endothelial cell proliferation and migration. VEGF antisense oligonucleotide treatment of HNSCC xenografts resulted in a significant tumor growth suppression. These results suggest that downmodulation of VEGF using antisense oligonucleotides may be a potential therapy for the inhibition of angiogenesis in HNSCC.     

Vascular endothelial growth factor overexpression by soft tissue sarcoma cells: implications for tumor growth, metastasis, and chemoresistance

To better elucidate the role of vascular endothelial growth factor (VEGF)(165) in soft tissue sarcoma (STS) growth, metastasis, and chemoresistance, we generated stably transfected human STS cell lines with VEGF(165) to study the effect of VEGF(165) on STS cells in vitro and the effect of culture medium from these cells on human umbilical vascular endothelial cells. Severe combined immunodeficient mice bearing xenografts of transfected cell lines were used to assess the effect of VEGF overexpression and the effect of VEGF receptor (VEGFR) 2 inhibition on STS growth, metastasis, and response to doxorubicin. VEGF(165)-transfected xenografts formed highly vascular tumors with shorter latency, accelerated growth, enhanced chemoresistance, and increased incidence of pulmonary metastases. Blockade of VEGFR2 signaling using DC101 anti-VEGFR2 monoclonal antibody enhanced doxorubicin chemoresponse; this combined biochemotherapy inhibited tumor growth and decreased pulmonary metastases without overt toxicity. Combined therapy reduced microvessel counts while increasing vessel maturation index. VEGF overexpression did not affect on the sarcoma cells per se; however, conditioned medium from VEGF transfectants caused increased endothelial cell proliferation, migration, and chemoresistance. Addition of DC101 induced endothelial cell sensitivity to doxorubicin and suppressed the activity of matrix metalloproteinases secreted by endothelial cells. We therefore conclude that VEGF is a critical determinant of STS growth and metastasis and that STS chemoresistance, in our model, is a process induced by the interplay between STS cells and tumor-associated endothelial cells. STS growth and metastasis can be interrupted by combined low-dose doxorubicin and anti-VEGFR2, a strategy that attacks STS-associated endothelial cells. In the future, such therapeutic approaches may be useful in treating STS before the development of clinically apparent metastases.     

Combination of anti‐angiogenic therapies reduces osteolysis and tumor burden in experimental breast cancer bone metastasis

The clinical efficacy of anti‐angiogenic monotherapies in metastatic breast cancer is less than originally anticipated, and it is not clear what the response of bone metastasis to anti‐angiogenic therapies is. Here, we examined the impact of neutralizing tumor‐derived vascular endothelial growth factor (VEGF) in animal models of subcutaneous tumor growth and bone metastasis formation. Silencing of VEGF expression (Sh‐VEGF) in osteotropic human MDA‐MB‐231/B02 breast cancer cells led to a substantial growth inhibition of subcutaneous Sh‐VEGF B02 tumor xenografts, as a result of reduced angiogenesis, when compared to that observed with animals bearing mock‐transfected (Sc‐VEGF) B02 tumors. However, there was scant evidence that either the silencing of tumor‐derived VEGF or the use of a VEGF‐neutralizing antibody (bevacizumab) affected B02 breast cancer bone metastasis progression in animals. We also examined the effect of vatalanib (a VEGF receptor tyrosine kinase inhibitor) in this mouse model of bone metastasis. However, vatalanib failed to inhibit bone metastasis caused by B02 breast cancer cells. In sharp contrast, vatalanib in combination with bevacizumab reduced not only bone destruction but also skeletal tumor growth in animals bearing breast cancer bone metastases, when compared with either agent alone. Thus, our study highlights the importance of targeting both the tumor compartment and the host tissue (i.e., skeleton) to efficiently block the development of bone metastasis. We believe this is a crucially important observation as the clinical benefit of anti‐angiogenic monotherapies in metastatic breast cancer is relatively modest.     

Characterization of epithelial senescence by serial analysis of gene expression: identification of genes potentially involved in prostate cancer

Evasion of cellular senescence is required for the immortal phenotype of tumor cells. The tumor suppressor genes p16(INK4A), pRb, and p53 have been implicated in the induction of cellular senescence. To identify additional genes and pathways involved in the regulation of senescence in prostate epithelial cells (PrECs), we performed serial analysis of gene expression (SAGE). The gene expression pattern of human PrECs arrested because of senescence was compared with the pattern of early passage cells arrested because of confluence. A total of 144,137 SAGE tags representing 25,645 unique mRNA species was collected and analyzed: 157 mRNAs (70 with known function) were up-regulated and 116 (65 with known function) were down-regulated significantly in senescent PrECs (P < 0.05; fold difference >2.5). The differential regulation of an exemplary set of genes during senescence was confirmed by quantitative real-time PCR in PrECs derived from three different donors. The results presented here provide the molecular basis of the characteristic changes in morphology and proliferation observed in senescent PrECs. Furthermore, the differentially expressed genes identified in this report will be instrumental in the further analysis of cellular senescence in PrECs and may lead to the identification of tumor suppressor genes and proto-oncogenes involved in the development of prostate cancer.     

Different sensitivities of senescent breast cancer cells to immune cell‐mediated cytotoxicity

Senescence is a state of growth arrest induced not only in normal cells but also in cancer cells by aging or stress, which triggers DNA damage. Despite growth suppression, senescent cancer cells promote tumor formation and recurrence by producing cytokines and growth factors; this state is designated as the senescence‐associated secretory phenotype. In this study, we examined the susceptibility of senescent human breast cancer cells to immune cell‐mediated cytotoxicity. Doxorubicin (DXR) treatment induced senescence in 2 human breast cancer cell lines, MDA‐MB‐231 and BT‐549, with the induction of γH2AX expression and increased expression of p21 or p16. Treatment with DXR also induced the expression of senescence‐associated β‐galactosidase and promoted the production of pro‐inflammatory cytokines. Importantly, DXR‐treated senescent MDA‐MB‐231 cells showed increased sensitivity to 2 types of immune cell‐mediated cytotoxicity: cytotoxicity of activated CD4⁺ T cells and Ab‐dependent cellular cytotoxicity by natural killer cells. This increased sensitivity to cytotoxicity was partially dependent on tumor necrosis factor‐related apoptosis‐inducing ligand and perforin, respectively. This increased sensitivity was not observed following treatment with the senescence‐inducing cyclin‐dependent kinase‐4/6 inhibitor, abemaciclib. In addition, treatment with DXR, but not abemaciclib, decreased the expression of antiapoptotic proteins in cancer cells. These results indicated that DXR and abemaciclib induced senescence in breast cancer cells, but that they differed in their sensitivity to immune cell‐mediated cytotoxicity. These findings could provide an indication for combining anticancer immunotherapy with chemotherapeutic drugs or molecular targeting drugs.     

Yeast-derived beta-glucan augments the therapeutic efficacy mediated by anti-vascular endothelial growth factor monoclonal antibody in human carcinoma xenograft models

PURPOSE: Bevacizumab is a recombinant IgG1 humanized monoclonal antibody against vascular endothelial growth factor (VEGF). Its proposed mechanism of action is independent of immune effector functions. Many human carcinomas not only secrete VEGF but also express membrane-bound VEGF. In addition, VEGF receptors are expressed on tumor cells. It is hypothesized that bevacizumab could bind membrane-bound VEGF or VEGF-VEGF receptor complexes on tumors, thereby initiating potential immunologic consequences. We previously showed that yeast-derived beta-glucan functions with antitumor antibodies that activate complement to recruit complement receptor 3-expressing leukocytes capable of mediating complement receptor 3-dependent cellular cytotoxicity of tumors opsonized with iC3b. In the current study, the therapeutic efficacy mediated by combining bevacizumab with yeast-derived beta-glucan was studied in human carcinoma xenograft models. EXPERIMENTAL DESIGN: Human tumor cell lines were screened for membrane-bound VEGF expression both in vitro and in vivo. Complement activation mediated by bevacizumab was examined. Tumor cell lines positive or negative for membrane-bound VEGF expression were implanted in severe combined immunodeficient mice to establish xenograft models. Tumor-bearing mice were treated with different regimens. Tumor regression and long-term survival were recorded. RESULTS: Human ovarian carcinoma SKOV-3 cells expressed membrane-bound VEGF both in vitro and in vivo. Bevacizumab was bound to membrane-bound VEGF, activated complement, and synergized with beta-glucan to elicit cellular cytotoxicity in vitro. In vivo study showed that beta-glucan could significantly augment the therapeutic efficacy mediated by bevacizumab. CONCLUSIONS: Yeast-derived beta-glucan can synergize with anti-VEGF monoclonal antibody bevacizumab for the treatment of cancer with membrane-bound VEGF expression.