eduzhai > Helth Sciences > Medical >

Pentoxifylline: an effective angiogenesis inhibitor by blocking STAT3 signaling in B16F10 melanoma

  • sky
  • (0) Download
  • 20211031
  • Save
https://www.eduzhai.net International Journal of Tumor Therapy 2013, 2(1): 1-9 DOI: 10.5923/j.ijtt.20130201.01 Pentoxifylline: A Potent Inhibitor of Angiogenesis via Blocking STAT3 Signaling in B16F10 Melanoma Dhumale Pratibha, Nikam Yuvraj, Gude Rajiv* Gude Lab. Advanced Centre for Treatment, Research and Education in Cancer (ACTREC) Tata M emorial Centre, Sector 22, Kharghar, Navi M umbai, 410 210, India Abstract Pento xifylline (PTX) has been shown to exert anti-metastatic activity in solid tumor. In the present study the underlying mo lecular mechanism of the anti- angiogenic activ ity of PTX was investigated in highly metastatic B16F10 melano ma. PTX suppressed the phosphorylation of Signal transducer activator of Transcription 3(STAT3) and its upstream kinases in B16f10 melano ma at nontoxic dosages. It also reduced the expression of HIF1α, VEGF (potent angiogenic factor), its receptors (VEGFR1, VEGFR2) and pro-inflammatory cytokines which play a significant role in induction of angiogenesis in solid tu mors. Moreover, PTX significantly suppressed the microvessel density around tumor induced in mice by injecting B16F10 cells intraderma lly on shaven ventral site. The inhibition in vasculature around tumor resulted in regression of tumor volume in PTX treated group of animals as compared to untreated group. These findings suggest that PTX exert anti-tu mor activity by inhibit ing angiogenesis via targeting the STAT3 signaling pathway in B16F10 melanoma Keywords Pento xifylline, STAT3, HIF1α, Angiogenesis, Melanoma 1. Introduction Melanoma is a comp lex genetic disease which is invasive by nature. Most deaths from melano ma are due to metastases that are resistant to conventional therapies. Metastasis is a sequential process which consists of interrelated steps such as adhesion, breakdown of basement memb rane, angiogenesis, motility and ho ming to distant target organ of tumor cells[1, 2]. Thus to target the spread of melano ma to distant organs it is necessary to restrict the disease at very early stages. One such early important stage is tumor angiogenesis[3]. It not only facilitates tumor growth but also provides an efficient route of exit for tu mor cells to leave the primary site and enter the blood stream Therefore a h ighly vascular tu mor is consider to have more ability to get metas tas ize[ 4]. Newly formed vasculature in solid tumors differs greatly fro m that found in normal t issue, typically displaying a broad range of structural & functional abnorma lit ies leading to the condition called as hypoxia[5]. It is regu lated by balance between mult iple pro and anti angiogenic factors such as VEGF (vas cular endothelial growth factor), Matrix metalloproteases (MMP2, MMP9), and pro -in flammatory cytokines etc. which are secreted either by tu mor cell itself or fro m host t issue in response to act ivat ion o f met abo lic * Corresponding author: rgude@actrec.gov.in (Gude Rajiv) Published online at https://www.eduzhai.net Copyright © 2013 Scientific & Academic Publishing. All Rights Reserved stress[6]. Theses pro-angiogenic factors further activate oncogenic cell survival pathways and one of them is the STAT3 (Signal Transduction & activation of Transcription 3) pathway, which contributes significantly to tumor progression. In response to cytokines and growth factors, STAT3 gets activated via phosphorylation through receptor associated kinases like JAK2 and Akt and gets dimerized and translocated to the nucleus, where it acts as transcription regulator of genes associated with angiogenesis and spread of cancer. In hypoxic conditions, STAT3 also stabilizes HIF1α, both of which regulate transcription of genes required for cell survival and prolife ration[9-11]. Thus there is a pressing need to identify a new agent which will target STAT3 and associated angiogenic factors, which are crucial for angiogenesis & metastasis in melanoma. Pentoxify lline (PTX) is a methyl xanthine derivative which is used in the treatment of peripheral vascular diseases and intermittent claudation. It enhances tumor sensitivity to both radiation and chemotherapeutic agents[12]. Earlier studies from our laboratory has shown that PTX causes cell cycle arrest in G0/ G1 phase at nontoxic dosages. It is a phosphodiestrase inhibitor which elevates cyclic AMP levels and impedes cancer cell migration and it also modulates surface expression of integrins and integrin mediated adhesion of B16F10 cells to extracellu lar matrix and lung homing[13]. However it remains unknown whether PTX affects any key targets involved in pathological tumor an g io g en es is . In the present study we investigated the molecular mechanis m of inhib ition of angiogenesis by PTX in B16F10 2 Dhumale Pratibha et al.: Pentoxifylline: A Potent Inhibitor of Angiogenesis via Blocking STAT3Signaling in B16F10 M elanoma melano ma and observed its effect on in vivo angiogenesis in C57BL/ 6 mice. We found that PTX inhib its STAT3 signaling and also demonstrates a decrease in expression of VEGF, its receptors and pro-inflammatory cytokines involved in angiogenesis. In vivo experiments in B16F10 melano ma showed inhibition of microvessel density around tumor and regression in tumor volu me on treatment with PTX. Our results suggest that PTX can be developed as a good candidate for anti-angiogenic therapy. 2. Materials and Methods 2.1. Cell Culture B16F10, a highly metastatic cell line was derived fro m C57BL/ 6 murine melano ma was purchased from national centre for cell science, Pune, India. The cell line was maintained as continuous culture in IMDM (Iscove’s minimu m dulbecco’s med iu m) supplemented with 10% fetal bovin serum (b iowest), 100 U/ ml penicilline& 100 µg/ ml streptomycin. Cells were gro wn in humidified at mosphere of 5% CO2 and at 95 % air at 37℃. IC50 value of pentoxifylline on B16F10 cells was found to be 39.2± 1.3 mM for 2 hr drug exposure of PTX, whereas at extended treatment of 24 hrs it is found to be 13.18± 1.8M m. For a ll our In Vitro experiment cells were treated with PTX at subtoxic dosage of 1 mM, 2 mM & 4 mM . C57BL/ 6 mice were purchased from Animal House, Advanced Centre for Treat ment Research Education in Cancer (A CTREC), Tata Memorial Centre, Kharghar, Navi Mumbai. A ll mice were used in accordance with the institutional guidelines of IA EC, A CTREC. Antibodies against STAT3, Phosphorylated STAT3 (tyr 705), JAK2, Phosphorylated JAK2, ERK1/2, Phospho ERK ½, Akt, PhosphoAkt, VEGF, HIF1α and CD31, were obtained from Santa Cruz Biotechnology. Antobodies against Tubulin was purchased from Sig ma Aldrich. 2.2. Western Blotting B16F10 cells were grown to 70-80 % confluency& treated with PTX for 24 hrs. The cells were scraped, follo wed by PTX treat ment and washed in ice cold PBS, and then whole-ce ll e xt racts were prepared by lysis buffer[50mM Tris, 1% Triton X-100, 150mM NaCl, 5mM EDTA, 10mM EGTA, 50 mM Na fluoride, 1mM Na orthovanadate, 1mM PMSF and protease inhibitor cocktail]. Lysates were spin at 10,000 rp m for 20 minutes to remove insoluble material. Supernatants were collected and kept at -80 C. Protein concentrations were determined with Bradford reagent. Whole cell lysates were resolved by 10% SDS-PA GE. After electrophoresis, the proteins were electro-transferred to PVDF membranes. Blocking was performed for 2 h at room temperature in blocking buffer consisting of 5% skim milk powder in TBST (Tris buffer saline, pH 7.4, 0.1% Tween-20). The memb rane was blotted with the relevant antibodies, and the proteins were detected by an enhanced chemilu minescence reagent (FEMTO). 2.3. Reverse Transcripti on-Pol ymerase Chain Reacti on (RT-PCR) Analysis RNA was ext racted by Trizo l (Sig ma Ald rich) method. cDNA preparation and PCR were perfo rmed using kit fro m Fermentas according to manufacturer’s instructions. Primers were manufactured by Sig ma Aldrich and had the following s equ en ces : Gene Prime r Se quence PC R condition ST AT3 Forward -5` TGGTGTCCA GTTTA CCACGA 3` Reverse- 5` TGGCGGCTTA GTGAA GAA GT 3` 94℃-3`(94℃-35S, 59℃-35S,72℃-35S- 30Cycles), 72-10` IL6 Forward- 5` ATGCTGGTGACAA CCACGGCC 3` 94℃-3`(94℃-35S, Reverse- 5` GGCATAACGCA CTA GGTTTGCCGA 3` 64℃-35S,72℃-35S-45 Cycles), 72-10` IFN Gamma Forward- 5` CCCA CA GGTCCA GCGCCAA G 3` Reverse- 5` TCGA GTGCTGTCTGGCCTGC 3` 94℃-3`(94℃-35S, 54℃-35S,72℃-35S-45 Cycles), 72-10` TNF alpha Forward-5` CTGCCGTCAA GA GA GCCCCTGC 3` Reverse- 5` GGGGGCTGGCTCTGTGA GGA 3` VEGF GAP DH Forward- 5` CA GGCTGCTGTAACGATGAA 3` Reverse- 5` TTTGA CCCTTTCCCTTTCCT 3` Forward 5-ATGGTGAA GGTCGGTGTGAACG-3' Reverse 5’-GTTGTCATGGATGACCTTGGCC-3' 94℃-3`( 94℃-35S, 62 ℃-35S,72℃ -35S-45 Cycles), 72-10` 94℃-3`( 94℃-35S, 57℃-35S,72℃-35S-30 Cycles), 72-10` 94℃- 3`( 94℃- 35S ,54℃- 35S, 72℃ -35S -30 Cycles), 72-10` International Journal of Tumor Therapy 2013, 2(1): 1-9 3 2.4. ELISA The B16F10 melano ma cell line was maintained in IMDM supplemented with 10% fetal bovine seru m and penicillin/streptomycin. To prepare supernatants from B16F10 cell line for ELISA, 3 ml o f fresh mediu m were added with or without PTX to established cell line when 60– 70% confluent in 60 mm petri dish and collected after 24 h. Supernatants were centrifuged at 1500 × g to remove particles, and aliquots were stored frozen at −80° until use in ELISA. Cell nu mber in the petri dish was determined to standardize the quantity of cytokine secreted per 106 tumor cells. VEGF level in cell culture supernatant was determined using an ELISA kit (A llied Life Science). According to manufacturer’s instruction and analyze using ELISA reader SPECRA MAX 190.The experiment was repeated three times . 2.5. Immunohistochemistry The localized tu mor along with blood vessels was fixed in buffered formaldehyde and embedded with paraffin. 5 µm sections of tumor tissue were processed to examine blood vasculature inside tumor tissue. Briefly , sections were deparaffin ised, washed with PBS, blocked with 5% BSA and incubated overnight at 4°C with primary antibody against specific endothelial cell marker CD31 .Fo llo wed by incubation with fluorescently conjugated secondary antibodies(anti goat conjugated with Alexa 568). Slides were then stained with DAPI for 1 minute in dark. Images were acquired at 10X using Trinocular Motorized Upright Research Microscope (ZEISS) 2.6. Determination of Effect of Pentoxifyllineon invivo Angiogenesis and on Tumor Development Angiogenesis was induced in three groups of C57BL/6 mice (5 mice/ group) by injecting B16-F10 melano ma cells (1 x 106) intradermally on shaven ventral skin surface of each mouse. Group I animals received Phosphate buffered saline (PBS) and served as control. Group II & III animals were treated with 20 mg/Kg & 40 mg/ Kg dose of pentoxifylline respectively by intraperitoneal route for 10 days after tumor injection. On Day 14 all an imals were sacrificed, the blood vessel density was observed & inhibition in blood vessel density was counted. 2.7. Statistical Analysis Data expressed as Mean + S.D o f three independent experiments. Statistical evaluation was done by applying student‘t’ test and One way ANOVA . whether PTX has any effect on STAT3 protein in B16F10 melano ma cells. We observed that PTX does not affect STAT3 at transcriptional or translational level but the active form of STAT3 i.e. phosphorylated form of STAT3 gets significantly inhib ited on PTX treat ment in a dose dependent manner (Fig. 1a, 1b ). As STAT3 is activated by upstream kinases such as JAK2, ERK 1/ 2 or AKT on ligand binding or stimulat ion of cytokines & growth factors[16-18], we analyzed activation (phosphorylation) of JAK2, ERK 1/2 and AKT in B16F10 cells on PTX treat ment by western blotting. PTX suppressed the phosphorylation of JAK2 and AKT (Fig. 1c, 1d). A similar inhibitory effect was observed on ERK 1/2 albeit at a higher concentration i.e. at 4 mM (Fig 1e). These results indicate that PTX inhibits the activation of STAT3 by inhibit ing the activation of its upstream kinases. 3.2. PTX Modul ates the Expression of Cytokines Regulated by STAT3 which are Invol ved i n Ang iog enes is Cytokines play an important role in tumo r development & angiogenesis[19]. We carried out studies to observe the effect of PTX on pro-inflammatory cytokines, which are mainly governed by STAT3, in B16F10 melanoma such as IL6, TNFα and IFN γ by RT PCR[20,21]. The results showed that TNFα, IFNγ are inhib ited significantly by PTX treatment in dose dependent manner. (Fig. 2) However no effect was observed on IL6 expression. 3.3. PTX Inhi bi ts Expression of HIF1 α & VEGF Hypoxia is one of the many conditions which induce the process of angiogenesis in solid tumors. HIF1α has been shown to carry out this induction when cancer cells reach a hypoxic state. STAT3 modulates the stability & activity of HIF1α and in turn enhances the expression of genes involved in angiogenesis & cell survival. STAT3 also interacts with HIF1α and gets recruited on VEGF pro moter region[22]. To determine the effect of PTX on HIF1α we checked the protein levels of HIF1α by western blot. We observed that the expression of HIF1 α was strongly inhibited by PTX at 1mm dose and further at higher doses (Fig.3a). As HIF1α, along with STAT3, acts as a transcriptional factor for expression of VEGF, we checked the effect of PTX on VEGF at mRNA & protein level by RT PCR & western blot respectively. We found a dose dependent decrease in VEGF expression at both transcriptional & translational level (Fig. 3 b, c). To quantify inhibit ion in VEGF secretion in the culture supernatant of B16F10 cells, ELISA was performed after incubating the cells with PTX. Th is inhibit ion was approximately 80% in 2 mM & 90 % in 4 mM (Fig. 3d). 3. Results 3.1. PTX Suppress STAT3 Signaling Pathway STAT3 is imp licated in cell survival, tumo r develop ment, angiogenesis and metastasis of melano ma[15]. We examined 3.4. PTX Inhi bi ts Expression of VEGF Receptors on B16F10 Cells Coexpression of VEGF & VEGF receptors such as Flk 1 (VEGFR1), KDR (VEGFR2) on tumor cell imp lies that autocrine loops exist between VEGF and VEGFR b inding. VEGF acts on tumor cells itself and further induces 4 Dhumale Pratibha et al.: Pentoxifylline: A Potent Inhibitor of Angiogenesis via Blocking STAT3Signaling in B16F10 M elanoma proliferative and angiogenic effect in solid tu mors[23,24]. angiogenesis. We found that expression of both receptors We, therefore, studied the effect of PTX on VEGF receptors significantly got inhibited by PTX treat ment in a dose such as Flk 1, & KDR wh ich are main receptors of VEGF in dependent manner in B16F10 cells (Fig. 3e). Figure 1. Pentoxifylline (PTX) suppress ST AT3 signaling – B16F10 cells were treated with 1mM, 2mM and 4mM dose of PT X for 24hrs. a) RT -PCR analysis of STAT3 mRNA expression. Whole cell lysates were subjected to western blotting to determine level of b) STAT3 and phosphor ST AT3 c) JAK2 and Phospho JAK2, d) Akt andPhosphoAkt, e) ERk1/2 and Phospho ERK1/2 Figure 2. PTX modulates the expression of cytokines regulated by ST AT3 which are involved in angiogenesis- B16 F10 cells were treated with PTX at dose of 1mM, 2mM and 4mM for 24hrs and mRNA expression of cytokines was studied a) IFN b)TNF α and c) IL6. The mRNA level was normalized in all t he cases t o GAPDH levels International Journal of Tumor Therapy 2013, 2(1): 1-9 5 Figure 3. PTX inhibits expression of HIF1α & VEGF- B16F10 cells were treated with PTX at 1 mM, 2mM and 4mM dose for 24 hrs. Whole cell lysates were subjected to western blotting to determine level of a) HIF1α, c) VEGF e) VEGFR1 and VEGFR2. b) RT PCR was performed to determine levels of VEGF mRNA after treatment of PTX at indicated concentrations. d) ELISA was performed to quantify VEGF levels in culture supernatants of B16F10 cells t reat ed wit h indicat ed concent rat ions of PTX for 24 hrs.(*p< 0.01 vs untreated control) Figure 4. PTX inhibits tumor induced microvessels- a) Photographs of skin sections of C57BL/6 mice implanted with B16F10 cells and treated with 20 mg/kg and40 mg/kg dose of PT Xfor 10 days. b) mean values of microvessels from mice of each group (n=5) with standard deviation are indicated. * p<0.01, **p<0.05 6 Dhumale Pratibha et al.: Pentoxifylline: A Potent Inhibitor of Angiogenesis via Blocking STAT3Signaling in B16F10 M elanoma Figure 5. PTX induces regression in tumor development- C57BL/6 mice implanted with B16F10 cells and treated with 20 mg/kg and 40 mg/kg dose of PTX for 10 days. a)Mean value of relative tumor volume (n=5) on day of sacrifice with standard deviation are indicated. * p<0.01, **p<0.05. b) CD31 staining in tumor tissue sections. Green color arrows indicate endothelial cells with CD31 marker stained with Alexa 568 3.5. PTX Inhi bi ts Tumor Induced Microvessels and Induces Regression in Tumor Devl opment The ability of PTX to inhibit newly formed vasculature around tumors was studied in intradermaly induced B16F10 melano ma tu mor on shaven ventral side of an imals[25]. We observed that the number o f tu mor induced capillaries was significantly reduced after PTX treat ment. Control animals had an average no. of 6 ± 1.63 capillaries whereas 4.25 ± 1.25 & 2.25 ± 0.95 average no. of capillaries were observed around the tumor of 20 mg/ kg & 40 mg/kg PTX treated group of animals (Fig. 5). To understand the effects of PTX on tumor development; the tumor volu me of localized tumor was measured by averaging the diameters using Verniercallipers. The tu mor vo lu me was expressed in mm3 using formula 0.4x a x b2. As shown in Figure 5, after 10 days treatment, the volu me of tu mors in PTX t reated groups was much sma ller than the control group. To further confirm the effect of PTX on blood vasculature inside the tumor tissue, we performed blood vessel staining of tumor tissue sections with endothelihal cell ma rker (CD31) antibody[26]. We observed that the average numbers of vessels in PTX treated groups were less than the control group. These results indicate that PTX not only inhibits angiogenesis but also tumor gro wth in vivo. 4. Discussion Melanoma is a malignancy of pig ment producing cells. Although melano ma at primary stage is curable through surgery, treatment of advance metastatic melano ma remains difficult and it is associated with poor prognosis. Despite world wide efforts in prevention, diagnosis, and treatment, cases of melano ma continue to rise. Extensive research and clin ical trials involving cytotoxic chemotherapeutic drugs, immunostimu latory agents such as interferon, Interleukin (IL2) both as single agent & in co mb ination reg imen have yielded low response rates against metastatic melanoma. Therefore it is necessary to target the disease before it attains its metastatic ability. As angiogenesis plays a critical role in growth & spread of melano ma, it is a potential target in melanoma therapy. Angiogenesis is characterized by proliferation of endothelial cells in tu mor t issue[27, 28]. In recent years several therapeutic approaches aimed at developing anti-angiogenic cytotoxic agents such as inhibitors of endothelial cells e.gAngiostat, GEM 220, Inhibitors of matrix metalloprotease COL3, Neovastat and modulators of upstream kinasees cetuxiamb, Herceptin etc have been tried[29, 30 ]. But the major problem associated with use of these anti-angiogenic International Journal of Tumor Therapy 2013, 2(1): 1-9 7 agents in melanoma therapy is that their use has shown complications in normal physiological function of kidney, heart as well as in wound healing process and also has revealed defects in fetus development[31. 32]. Thus there is a pressing need to develop an anti-angiogenic agent which will target abnormal mic rovessel density in tumor tissue, and will be less toxic causing no developmental or physiological disorders. One of the pro mising approaches in this regard is to investigate the anti-angiogenic potential of drugs that already have shown their efficacy in treat ment of other diseases. This will facilitate early imp lementation of that drug into clin ical setup as its pharmacokinetic data and side effects are already known. Pentoxify lline (PTX) is a theobromine derivative and its chemical name is 1-(5-o xohexy l) - 3, 7-dimethylxanthine. Clin ically this co mpound is used to treat a peripheral vascular disease and is commercially available in the name of Trental. PTX shows its anti proliferative, anti-adhesive, anti-metastatic properties on melano ma cell line at non toxic dosage[33-36]. Therefore PTX draws attention for its use in antimetastasis therapy. In the present study we report that PTX exerts its anti angiogenic activity by targeting STAT3 signaling in the highly metastatic B16F10 melanoma model. STAT3 is constitutively activated at 50 to 90% frequency in diverse forms of cancer including melano ma. It is a cytoplasmic protein wh ich gets activated by upstream kinases via ligand binding or through cytokines or growth factors[37]. A critical ro le of STAT3 activation in tu mor cell survival, proliferation, angiogensis, metastasis and immune evasion has been well demonstrated. STAT3 contributes to tumor cell survival by regulating the expression of genes that are involved in cell survival[38]. STAT3 pro motes metastasis &angiogensis by inducing expression of metastatic genes, MMPs, serine protease uPAand potent angiogenic genes, bFGF and VEGF. STAT3 signaling in dendritic cells leads to immune tolerance[39, 40]. STAT3 is, thus, an attractive target for cancer therapy including advanced metastatic melano ma. Our data demonstrate that PTX significantly inhibits the activation of STAT3 in the highly metastatic B16F10 melano ma. The inhib itory effect is associated with inhibition in activation of the upstream kinase JAK2. Janus associated kinase (JA K) has tyrosine kinase activity, which binds to cytokine receptors and gets activated and further triggers the activation of STAT3. It has been reported by other investigatorsthat inhibition in activation of JAK2/STAT3 signaling pathway results in inhib ition of tumo r gro wth[42]. ERK 1/ 2 and A kt are found to be constitutively activated in melano ma and play an important role in tu mor develop ment. Inhibition in activation of ERK 1/2 or PI 3/Akt kinase induces cell death[43]. Both of these mo lecules are involved in activation of STAT3. In our study we show that PTX inhibits the phosphorylation of Akt in a dose dependent manner and a similar inhib itory effect on pERK1/ 2 is observed at higher concentration of PTX i.e. at 4 mM . This might be one of the ways through which the activity of STAT3 gets inhibited by PTX; but the possibility of other ways of inhibit ion of STAT3 exists. Altered levels of pro-inflammatory cytokines which are also pro-angiogenic factors are observed in various forms of cancer including melanoma. The cytokines such as IL-6, IFNγ and TNF α act as autocrine growth factors; they are secreted by tumor cells and in turn they activate cell survival by activating STAT3 signaling[43-45]. We observed a significant reduction in exp ression of IFNγ and TNF α at the transcriptional level on PTX treat ment. However no effect was observed on IL6. It is possible that some other transcription factor, along with STAT3, is involved in synthesis of IL6 in B16F10 melano ma. Recently, Niu and colleagues reported that constitutive activity of STAT3 up-regulates VEGF exp ression and tumor angiogenesis[46]. In hypoxic condition of solid tumors STAT3 stabilizes HIF 1α through delaying protein ubiquitination& accelerat ing protein synthesis. HIF 1 alpha is also regulated at the level o f protein initiat ion by activation of Akt . HIF 1α along with STAT3 acts as a transcription factor for VEGF exp ression in tu mor cells[47, 48]. Our results suggest that protein levels of HIF 1α get significantly decreased on PTX t reat ment in B16F10 melanoma. Consistently PTX treat ment also causes a remarkab le decrease in expression of VEGF at transcriptional & translational level. Co-exp ression of VEGF and its receptors on the tumor cell imp lies that VEGF may directly function on tumor cells themselves by enhancing downstream signaling such as STAT3 signaling[49, 50]. PTX treat ment shows a remarkable decrease in the expression of VEGF receptors (VEGFR1, VEGFR2). The exact molecular mechanis m o f this inhibit ion on VEGF receptors is still unclear. We plan to study this in the future. The above results indicate that PTX can exert its anti-angiogenic effect in B16F10 melano ma by inhibit ing expression of VEGF & its recep to rs . To confirm the anti-angiogenic effect of PTX in vivo we injected B16F10 ce lls intradermaly on shaven ventral side of C57BL/ 6 mice. In this in vivo angiogenesis assay we found that PTX inhib its tumor induced new blood vessel formation without any side effects in C57BL/ 6 mice in a dose dependent manner. This effect was observed with no toxicity in mice wh ich fu rther gives advantage to using PTX in anti-angiogenic therapy of cancer treatment. In summary our results show that PTX downregulates the activation of STAT3 v ia inhibit ing the activation of its upstream kinases. PTX t reatment also inhibits HIF1α. Inhibition in activation of STAT3and HIF1α further decreases the expression of the gene VEGF which is involved in angiogenesis. Inhibition in angiogenesis in vivo corroborates our theory that PTX can be used as a potent anti-angiogenic agent. Thus PTX treat ment could be beneficial in melano ma therapy and should be taken in serious consideration for its imp lementation as an anti-angiogenic agent for adjuvant therapy. 8 Dhumale Pratibha et al.: Pentoxifylline: A Potent Inhibitor of Angiogenesis via Blocking STAT3Signaling in B16F10 M elanoma ACKNOWLEDGEMENTS The Authors would like to e xpress their gratitude to Indian council of medica l research (ICM R), New De lhi for funding. REFERENCES [1] Zetter, B. R. (1998). "Angiogenesis and tumor metastasis." Annu Rev M ed49: 407-424. [15] Kusaba, T., T. Nakayama, et al. (2006). "Activation of STAT3 is a marker of poor prognosis in human colorectal cancer." Oncol Rep15(6): 1445-1451. [16] Kuroki, M . and J. T. O'Flaherty (1999). "Extracellular signal-regulated protein kinase (ERK)-dependent and ERK-independent pathways target STAT3 on serine-727 in human neutrophils stimulated by chemotactic factors and cytokines." Biochem J341 ( Pt 3): 691-696. [17] Lin, N., Y. M oroi, et al. (2007). "Significance of the expression of phosphorylated-STAT3, -Akt, and -ERK1/2 in several tumors of the epidermis." J Dermatol Sci48(1): 71-73. [2] Neitzel, L. T., C. D. Neitzel, et al. (1999). "Angiogenesis [18] Plaza-M enacho, I., T. van der Sluis, et al. (2007). correlates with metastasis in melanoma." Ann SurgOncol6(1): "Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by 70-74. familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for [3] M acchiarini, P., G. Fontanini, et al. (1994). "Angiogenesis: an c-fos promoter activation, cell mitogenicity, and indicator of metastasis in non-small cell lung cancer invading transformation." J Biol Chem282(9): 6415-6424. the thoracic inlet." Ann Thorac Surg57(6): 1534-1539. [19] Bode, J. G., U. Albrecht, et al. (2011). "Hepatic acute phase [4] Bhat, T. A. and R. P. Singh (2008). "Tumor angiogenesis--a proteins - Regulation by IL-6- and IL-1-type cytokines potential target in cancer chemoprevention." Food Chem involving STAT3 and its crosstalk with NF-kappaB-depende Toxicol46(4): 1334-1345. nt signaling." Eur J Cell Biol. [5] Hoekman, K. and H. M . Pinedo (2004). "Angiogenesis: a potential target for therapy of soft tissue sarcomas." Cancer Treat Res120: 169-180. [6] Hendriksen, E. M ., P. N. Span, et al. (2009). "Angiogenesis, hypoxia and VEGF expression during tumour growth in a human xenograft tumour model." M icrovasc Res77(2): 96-103. [20] Sunila, E. S. and G. Kuttan (2006). "Piper longum inhibits VEGF and proinflammatory cytokines and tumor-induced angiogenesis in C57BL/6 mice." Int Immunopharmacol6(5): 733-741. [21] Aleffi S, et al. (2005) Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells. Hepatology 426: 1339-48 [7] Zhao, M ., F. H. Gao, et al. (2011). "JAK2/STAT3 signaling [22] Jung, J. E., H. G. Lee, et al. (2005). "STAT3 is a potential pathway activation mediates tumor angiogenesis by modulator of HIF-1-mediated VEGF expression in human upregulation of VEGF and bFGF in non-small-cell lung renal carcinoma cells." FASEB J19(10): 1296-1298. cancer." Lung Cancer73(3): 366-374. [23] Aesoy, R., B. C. Sanchez, et al. (2008). "An autocrine [8] Li, Y., P. Kundu, et al. (2012). "Gut M icrobiota accelerate VEGF/VEGFR2 and p38 signaling loop confers resistance to Tumor Growth via c-Jun and STAT3 Phosphorylation in 4-hydroxytamoxifen in M CF-7 breast cancer cells." M ol APCM in/+ M ice." Carcinogenesis. Cancer Res6(10): 1630-1638 [9] M ora, L. B., R. Buettner, et al. (2002). "Constitutive activation of Stat3 in human prostate tumors and cell lines: direct inhibition of Stat3 signaling induces apoptosis of prostate cancer cells." Cancer Res62(22): 6659-6666. [10] Gray, M . J., J. Zhang, et al. (2005). "HIF-1alpha, STAT3, CBP/p300 and Ref-1/APE are components of a transcriptional complex that regulates Src-dependent hypoxia-induced expression of VEGF in pancreatic and prostate carcinomas." Oncogene24(19): 3110-3120. [11] Xu, Q., J. Briggs, et al. (2005). "Targeting Stat3 blocks both HIF-1 and VEGF expression induced by multiple oncogenic growth signaling pathways." Oncogene24(36): 5552-5560. [12] Gastpar, H. (1974). "The inhibition of cancer cell stickness by the methylxanthine derivative pentoxifylline (BL 191)." Thromb Res5(3): 277-289. [13] Gude, R. P., A. D. Ingle, et al. (1996). "Inhibition of lung homing of B16F10 by pentoxifylline, a microfilament depolymerizing agent." Cancer Lett106(2): 171-176. [14] Zeisberg, E. M ., S. Potenta, et al. (2007). "Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts." Cancer Res67(21): 10123-10128. [24] Chen H, et al. (2004) VEGF, VEGFRs expressions and activated STATs in ovarian epithelial carcinoma. Gynecol Oncol 94 3): 630-5 [25] M enon, L. G., A. D. Ingle, et al. (2002). "Tumor regression of B16F10 melanoma in vivo by prevention of neovascularization: study on theophylline." Cancer Biother Radiopharm17(2): 213-217. [26] Zhang, X., Y. Song, et al. (2011). "Indirubin inhibits tumor growth by antitumor angiogenesis via blocking VEGFR2mediated JAK/STAT3 signaling in endothelial cell." Int J Cancer129(10): 2502-2511. [27] Wu, D. H., L. Liu, et al. (2004). "Radiosensitization by pentoxifylline in human hepatoma cell line HepG2 and its mechanism." Di Yi Jun Yi Da Xue Xue Bao24(4): 382-385. [28] Ravi K. Amaravadi (2007), “Targeted Therapy forM etastatic M elanoma” Clinical Advances in Hematology & Oncology. [29] Joyce E. Rundhaug(2003) “M atrix M etalloproteinases, Angiogenesis, and Cancer”Clin Cancer Res , 551-554. [30] Giampaolo Tortora1,DavideM elisi1 ,Fortunato Cia (2009) “Angiogenesis: A Target for Cancer Therapy” Current Pharmaceutical Design, 10, 11-26. International Journal of Tumor Therapy 2013, 2(1): 1-9 9 [31] Han-Chung Wu,et al.(2008) “Anti-Angiogenic Therapeutic Drugs for Treatment of Human Cancer”Journal of Cancer M olecules 4(2): 37-45, 2008 [32] Nalluri SR, chu D, Keresztes R, Zhu X, Wu S. (2008) “Risk of venous thromboembolism in the angiogenesis inhibitor bevacizuamab in cancer patients” JAMA 300(19) 2277-2285. [33] Dua, P. and R. P. Gude (2008). "Pentoxifylline impedes migration in B16F10 melanoma by modulating Rho GTPase activity and actin organisation." Eur J Cancer44(11): 1587-1595. [41] Zhao, M ., F. H. Gao, et al. (2011). "JAK2/STAT3 signaling pathway activation mediates tumor angiogenesis by upregulation of VEGF and bFGF in non-small-cell lung cancer." Lung Cancer73(3): 366-374. [42] Lin, N., Y. M oroi, et al. (2007). "Significance of the expression of phosphorylated-STAT3, -Akt, and -ERK1/2 in several tumors of the epidermis." J Dermatol Sci48(1): 71-73. [43] Chen, Z., P. S. M alhotra, et al. (1999). "Expression of proinflammatory and proangiogenic cytokines in patients with head and neck cancer." Clin Cancer Res5(6): 1369-1379. [34] Gude RP, et al. (2001) Inhibition of endothelial cell proliferation and tumor-induced angiogenesis by pentoxifyll ine. J Cancer Res ClinOncol 127 10): 625-30 [35] Goel, P. N. and R. P. Gude (2011). "Unravelling the antimetastatic potential of pentoxifylline, a methylxanthine derivative in human M DA-M B-231 breast cancer cells." M ol Cell Biochem358(1-2): 141-151. [36] Jain, M ., A. Ratheesh, et al. (2010). "Pentoxifylline inhibits integrin-mediated adherence of 12(S)-HETE and TNFalphaactivated B16F10 cells to fibronectin and endothelial cells." Chemotherapy56(1): 82-88. [37] Huang, S. (2007). "Regulation of metastases by signal transducer and activator of transcription 3 signaling pathway: clinical implications." Clin Cancer Res13(5): 1362-1366. [38] M imori, K., T. Fukagawa, et al. (2008). "A large-scale study of M T1-MMP as a marker for isolated tumor cells in peripheral blood and bone marrow in gastric cancer cases." Ann Surg Oncol15(10): 2934-2942. [39] Iwata-Kajihara, T., H. Sumimoto, et al. (2011). "Enhanced cancer immunotherapy using STAT3-depleted dendritic cells with high Th1-inducing ability and resistance to cancer cell-derived inhibitory factors." J Immunol187(1): 27-36. [40] Alshamsan, A., A. Haddadi, et al. (2010). "STAT3 Silencing in Dendritic Cells by siRNA Polyplexes Encapsulated in PLGA Nanoparticles for the M odulation of Anticancer Immune Response." M ol Pharm. [44] Snyder, M ., X. Y. Huang, et al. (2011). "Signal transducers and activators of transcription 3 (STAT3) directly regulates cytokine-induced fascin expression and is required for breast cancer cell migration." J Biol Chem286(45): 38886-38893. [45] Shu, M ., Y. Zhou, et al. (2011). "Activation of a pro-survival pathway IL-6/JAK2/STAT3 contributes to glial fibrillary acidic protein induction during the cholera toxin-induced differentiation of C6 malignant glioma cells." M ol Oncol5(3): 265-272. [46] Niu, Z. B., C. L. Wang, et al. (2007). "Expression of Stat3, HIF-1alpha and VEGF in Wilms' tumor." Zhongguo Dang Dai Er Ke Za Zhi9(5): 461-464. [47] Shin, J., H. J. Lee, et al. (2011). "Suppression of STAT3 and HIF-1 alpha mediates anti-angiogenic activity of betulinic acid in hypoxic PC-3 prostate cancer cells." PLoS One6(6): e21492. [48] Zhang, Q., C. K. Oh, et al. (2006). "Hypoxia-induced HIF-1 alpha accumulation is augmented in a co-culture of keloid fibroblasts and human mast cells : involvement of ERK1/2 and PI-3K/Akt." Exp Cell Res312(2): 145-155. [49] Waldner, M . J., S. Wirtz, et al. (2010). "VEGF receptor signaling links inflammat ion and tumorigenes is in colitisassociated cancer." J Exp M ed207(13): 2855-2868. [50] Olsson, A. K., A. Dimberg, et al. (2006). "VEGF receptor signalling - in control of vascular function." Nat Rev M ol Cell Biol7(5): 359-371.

... pages left unread,continue reading

Document pages: 9 pages

Please select stars to rate!

         

0 comments Sign in to leave a comment.

    Data loading, please wait...
×