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Inositol Hexaphosphate (IP6) Inhibits Cellular Proliferation In Melanoma
Journal of Surgical Research 133, 3– 6 (2006) doi:10.1016/j.jss.2006.02.023
Inositol Hexaphosphate (IP6) Inhibits Cellular Proliferation In Melanoma 1 Irfan Rizvi, M.D., Dale R. Riggs, Barbara J. Jackson, Alex Ng, Cynthia Cunningham, Ph.D., and David W. Mcfadden, M.D., F.A.C.S.2 Department of Surgery, Robert C. Byrd Health Science Center, West Virginia University, Morgantown, West Virginia Submitted for publication January 9, 2006
Key Words: melanoma; inositol hexaphosphate; apoptosis; VEGF.
Background. Inositol Hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate found in food sources high in fiber content. We have previously reported IP6 to have significant inhibitory effects against pancreatic cancer in vitro. We hypothesized that the IP6 would significantly inhibit cell growth of cutaneous melanoma in vitro. Materials and methods. The melanoma line HTB68 was cultured using standard techniques and treated with IP6 at doses ranging from 0.2 to 1.0 mM/well. Cell viability was measured by MTT at 72 h. VEGF production was measured in the cell supernatants by ELISA. Apoptosis was evaluated by Annexin V-FITC and results calculated using FACS analysis. Statistical analysis was performed by ANOVA. Results. Significant reductions (P < 0.001) in cellular proliferation were observed with IP6. Overall, IP6 exhibited a mean inhibition of cell growth of 52.1 ⴞ 11.5% (range, 1.6 – 83.0%) at 72 h of incubation. VEGF production was significantly reduced (P < 0.001) by the addition of IP6 (7.5 pg/ml) compared to control (40.9 pg/ml). IP6 significantly increased (P ⴝ 0.029) late apoptosis from 5.3 to 7.0% gated events. No changes in necrosis or early apoptosis were observed. Conclusions. Adjuvant treatment of melanoma continues to challenge clinicians and patients. Our findings that IP6 significantly decreased cellular growth, VEGF production and increased late apoptosis in melanoma suggest its potential therapeutic value. Further in vivo studies are planned to evaluate safety and clinical utility of this agent. © 2006 Elsevier Inc. All rights reserved.
INTRODUCTION
The incidence of melanoma continues to grow, with an estimated 59,580 new cases in 2005. It is estimated that there would be 7,770 mortalities directly because of melanoma in 2005 [1]. After the diagnosis of melanoma, the first course of treatment for locally advanced and/or systemic disease is surgical resection, followed by immunotherapy, chemotherapy and/or radiation therapy [2, 3]. While these treatments are effective in many patients, not all patients will respond and many will have recurrence and or progression of the melanoma. For these patients, new and potentially more effective forms of therapy are continually being investigated. Inositol Hexaphosphate (IP6) is a complex carbohydrate that is a naturally occurring compound found in grains, cereals, nuts, and foods that are high in fiber content. IP6 has been reported to have significant beneficial properties in a number of investigational cancer models, including cancers of the prostate [4, 5], breast [6], colon [7], and pancreas [8]. There has been no reported investigation or evaluation of the effects of IP6 on melanoma. In a continuing effort to evaluate safer and more effective forms of therapy, we investigated the in vitro effects of IP6 on melanoma. MATERIALS AND METHODS
1
Presented at the 1st Annual Academic Surgical Congress, February 7–11, 2006, at the Hyatt Regency Manchester Grand in San Diego, CA. 2 To whom correspondence and reprint requests should be addressed at Department of Surgery, WVU, #1 Medical Center Drive, PO Box 9238, Morgantown, WV 26506. E-mail: dmcfadden@hsc. wvu.edu.
Inositol Hexaphosphate (IP6) IP6 (dodecasodium salt) derived from corn (Sigma Catalogue #P8810) was purchased from Sigma Chemical Co. (St. Louis, MO). IP6 was solubilized in tissue culture media to the desired concentrations of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mM/well.
3
0022-4804/06 $32.00 © 2006 Elsevier Inc. All rights reserved.
4
JOURNAL OF SURGICAL RESEARCH: VOL. 133, NO. 1, JUNE 1, 2006 bind to the antibody. The microplate was then washed to release any unbound substances and an enzyme-lined polyclonal antibody specific for VEGF was added to each well. After incubation, a wash buffer was used to remove any unbound antibody-enzyme reagent and a substrate solution was then added for color development. Once the color development was complete, the plate was read at 540 nm.
Statistical Analysis Determination of statistical significance was performed by analysis of variance [10]. Post hoc comparison of individual concentration means with the control was completed using the Tukey-Kramer Multiple Comparison test [11]. All data are reported as means and standard errors of the means.
RESULTS Reduction of Cellular Proliferation
FIG. 1. Titration of the effects IP6 (corn) on melanoma in vitro evaluated by the MTT assay 72 h incubation.
Cell Culture and Reagents The melanoma cell line HTB68 was purchased from American Type Tissue Collection (ATCC, Manassas, VA). The cell line was cultured and maintained as a monolayer in the preferred media and maintained in monolayer culture at 37°C in humidified air with 5% CO 2. Cells were plated in sterile 96-well plates at 1 ⫻ 105 cells/ml and incubated for 72 h in the presence of IP6 at concentrations listed above, while an equal volume of vehicle was added representing the controls.
MTT Assay The MTT colorimetric assay was performed to detect tumor cell viability after 72 h of incubation. MTT, a tetrazolium dye (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue, Sigma, St. Louis, MO) was added to the each well as described previously [9]. Plates were incubated in the presence of MTT dye for 4 h. Mitochondrial dehydrogenase activity reduced the yellow MTT dye to a purple formazan, which was then solubilized with acidified isopropanol and absorbance was read at 570 nm on an ELISA plate reader.
Significant reductions in cellular proliferation were observed in the HTB68 melanoma cell line by the addition of 0.3 to 1.0 mM/well IP6 (P ⬍ 0.05). The reduction in cellular proliferation ranged from 12.3% ⫾ 4.1 with 0.4 mM/well to 83.0% ⫾ 0.4 with 0.8 mM/well of IP6. Overall, a reduction in cellular proliferation of 52.1% ⫾ 11.5 was observed over doses tested (Fig. 1, P ⬍ 0.001). A dose of 0.35 mM/well was used in subsequent experiments evaluating the effects of IP6 on apoptosis, necrosis, and VEGF production. This dose was chosen based upon its reduction of cellular proliferation observed in the MTT assay (approximately 20%). Evaluation Apoptosis and Necrosis
Late apoptosis was significantly increased by the addition of 0.35 mM/well of IP6 (7.0 ⫾ 0.17) compared to the control (5.3 ⫾ 0.23, P ⫽ 0.029; Fig. 2). No change was noted in either early apoptosis (4.7 ⫾ 0.23) or necro-
Measurement of Apoptosis Annexin V-FITC Apoptosis Detection Kit (Biovision Inc., Mountain View, CA) was used to detect early and late apoptotic activity after 18 h of incubation. After treatment wit 0.35 mM/well of IP6, cells (1 ⫻ 106) were collected and resuspended in binding buffer, and Annexin V-FITC and Propidium Iodide (PI) were added to each sample and incubated in the dark for 5 min. Annexin V-FITC binding was determined by flow cytometry (Ex ⫽ 488 nm; Em ⫽ 530 nm) using FITC signal detector (FL1) and PI staining by the phycoerythrin emission signal detector (FL2).
Human VEGF Quantikine ELISA Kit A commercial ELISA kit (R&D Systems, Minneapolis, MN) was used to detect VEGF (Vascular Endothelial Growth Factor) Production (pg/ml) after 72 h of incubation. After the cells were treated with IP6, the supernatants were collected for quanitification of VEGF production. Cell culture supernatants, standards, or control samples were added in 200 l to a pre-coated microplate containing a monoclonal antibody specific for VEGF. VEGF present in the samples will
FIG. 2. Annexin V determination of apoptotic activity in the HTB68 melanoma in vitro.
RIZVI ET AL.: EFFECTS OF INOSITOL HEXAPHOSPHATE ON MELANOMA
FIG. 3.
VEGF determination of HTB68 melanoma in vitro.
sis (0.1 ⫾ 0.01) by the addition of IP6 in vitro when compared to control (7.2 ⫾ 1.04 and 0.3 ⫾ 0.02, N.S.). Evaluation of In Vitro VEGF Production
IP6 0.35 mM/well significantly reduced the VEGF levels in tissue culture cell supernatants of the HTB68 cells when compared to controls (7.5 ⫾ 1.5 9pg/ml versus 40.9 ⫾ 9. pg/ml, P ⬍ 0.001, Fig. 3). DISCUSSION
Melanoma is a cancerous tumor of melanocytes, with the majority being diagnosed in adults. Early warning signs of melanoma include darkening or change in color, irritation, or itching, and or increased size of existing nevi. Should any of these signs be discovered, a biopsy of the area is indicated. The first line of treatment after detection of melanoma, surgical resection, is a highly effective form of treatment for those patients diagnosed with early stage melanoma. The majority of these patients will not require any additional form of treatment other than surgical resection. However, for patients with advanced stage melanoma (stage III and IV) that has metastasized to the lymph nodes or distant organs, surgical resection alone is not an option. These patients require adjuvant treatment with chemotherapy, immunotherapy, tumor vaccines, or other experimental treatment protocols. However, these forms of treatment are expensive, have substantial co-morbidities and produce marginal benefit in a great number of patients. For this reason the search for more effective and less toxic treatment options for patients suffering from melanoma continues. IP6 is a complex carbohydrate that is a found in grains, cereals, nuts, and foods high in fiber content. IP6 has been reported to significantly decrease the growth of cancers of the prostate [4, 5] breast [6], and
5
colon [7]. We have previously evaluated the in vitro effects of IP6 on human pancreatic cancer [8]. Significant reductions in cell proliferation was observed after 72 h of treatment with IP6 in all doses tested (P ⬍ 0.001). Vucenik et al. [12], also studied the effects of IP6 (dodecasodium salt derived from rice) on cell growth in the bovine aortic endothelial cells (BAECs) and VEGF production in the human liver cancer cell line (HepG2). They observed that IP6 inhibited cellular growth in a dose-dependent manner on day 3 (IC 50 ⫽ 0.74 mM), but lower doses of IP6 (0.1 and 0.3 mM) had no effect on cell growth when compared to the control. However, significant growth inhibition was observed with 0.6 mM of IP6 (84% on day 3 and 62% on day 5). In our current study, significant growth inhibition was observed in the HTB68 melanoma cell line with doses ranging from 0.3 to 1.0 mM/well of IP6 (range of 12.3% to 83.0%) at 72 h of treatment. This data indicates that IP6 may have the potential to become an effective adjuvant treatment for melanoma. Among the reported mechanisms by which IP6 exerts its anti-proliferative effect are through regulation of apoptosis and angiogenesis. Argarwal et al. [5] investigated whether IP6 would inhibit NF-kappa B, which is active in advanced and androgen-independent human prostate cancer cells (DU145), as well as its effects on proliferation and apoptosis. Their group found that when the DU145 cells were treated with doses of 1 and 2 mM of IP6, cellular proliferation was strongly inhibited and apoptotic activity was induced. In other work, IP6 has been shown to significantly increase caspase-3 activity in an experimental mouse prostate model [13]. Sharma et al. evaluated the effects of IP6 in the transgenic adenocarcinoma of mouse prostate (TRAMP-C1) cell line. The TRAMP-C1 cells were treated with various doses of IP6 (ranging from 0.5 to 4.0 mM) for 24 – 72 h. Their results included 6 to 35% cell death, in a dose and time-dependent manner. They also showed a significant induction (six-fold) in caspase-3 activity when compared to control. In the current study, apoptotic activity was measured after 18 h of incubation with 0.35 mM IP6 and no changes were observed in early apoptosis or necrosis; however, late apoptosis was significantly increased when compared to control (P ⫽ 0.029). This data are also supported by our previous work in that apoptotic activity was significantly increased by IP6 in both MiaPaca and PANC-1 human pancreatic cancer cells [8]. VEGF production was also measured to evaluate the anti-angiogenic properties of IP6. VEGF production was significantly reduced with 0.35 mM of IP6 by 81.7% (7.5 ⫾ 0.8 pg/ml) when compared to the control (40.9 ⫾ 4.8 pg/ml) (P ⬍ 0.001). Vucenik et al., also examined the effects of IP6 on VEGF production in HepG2 cells in their two series of experiments. In the first experi-
6
JOURNAL OF SURGICAL RESEARCH: VOL. 133, NO. 1, JUNE 1, 2006
ment, they measured VEGF protein in HepG2 cells treated with increasing concentrations of IP6 (0.5 mM, 1.0 mM, and 2.0 mM), and aliquots of media were removed at 1, 3, 6, and 24 h after incubation. In the second experiment, VEGF protein was measured in HepG2 cells pre-treated with several doses of IP6 (0.5 mM, 1.0 mM, and 2.0 mM) for 24 h. After 24 h, the media was replaced with serum free medium and aliquots were then collected at 3, 6, and 24 h. The researchers found that in the first experiment, IP6 decreased VEGF by approximately 50% after 24 h, in a concentrationdependent manner. However, when the cells were pretreated with various doses of IP6, VEGF production was reduced by 79% at a dose of 1.0 mM, while an 86% reduction of VEGF was observed with 2.0 mM [12]. There have been no reports on the effectiveness of IP6 in melanoma in vivo or in clinical use. However, IP6 has been shown to reduce significantly the development of skin cancer in experimental animal models. Ishikawa et al. studied the effects of 2% IP6 in drinking water on 7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer in mice. Animals received IP6 in one of two schedules. The first was the initiation period (3 weeks before tumor induction) and the second was the promotion phase (19 weeks post-tumor induction). The animals receiving IP6 during the initiation period exhibited a 50% reduction in the number of tumors per animal and in the overall incidence of tumor. In contrast, no such inhibition was observed in the group of animals receiving IP6 in the promotion phase [14]. Gupta et al. also investigated the effect of IP6 as a topical agent on DMBA-induced mouse skin tumors. Their results showed that IP6, administered topically, significantly inhibited skin tumor development, presumably by affecting proliferation, differentiation, and by inducing apoptosis [15].
clinical trials to determine the clinical efficacy of IP6 in the management of this disease. REFERENCES 1. 2.
3. 4.
5.
6.
7.
8.
9.
10. 11. 12.
13.
CONCLUSIONS
In summary, we have shown for the first time that IP6 significantly inhibits the in vitro growth of melanoma. Anti-angiogenic and pro-apoptotic mechanisms were demonstrated. The results reported here will lead to in vivo studies, and possibly subsequent phase II
14.
15.
Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10. McGillis ST, Fein H. Topical treatment strategies for nonmelanoma skin cancer and precursor lesions. Semin Cutan Med Surg 2004;23:174. Villa AM, Berman B. Immunomodulators for skin cancer. J Drugs Dermatol 2004;3:533. Singh RP, Sharma G, Mallikarjuna GU, Dhanalakshmi S, Agarwal C, Agarwal R. In vivo suppression of hormonerefractory prostate cancer growth by inositol hexaphosphate: Induction of insulin-like growth factor binding protein-3 and inhibition of vascular endothelial growth factor. Clin Cancer Res 2004;10:244. Agarwal C, Dhanalakshmi S, Singh RP, Agarwal R. Inositol hexaphosphate inhibits constitutive activation of NF- kappa B in androgen-independent human prostate carcinoma DU145 cells. Anticancer Res 2003;23:3855. Tantivejkul K, Vucenik I, Shamsuddin AM. Inositol hexaphosphate (IP6) inhibits key events of cancer metastasis: II. Effects on integrins and focal adhesions. Anticancer Res 2003;23:3681. Shamsuddin AM, Elsayed AM, Ullah A. Suppression of large intestinal cancer in F344 rats by inositol hexaphosphate. Carcinogenesis 1988;9:577. Somasundar P, Riggs DR, Jackson BJ, Cunningham C, VonaDavis L, McFadden DW. Inositol hexaphosphate (IP6): A novel treatment for pancreatic cancer. J Surg Res 2005;126:199. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methodsi 1983;65:55. Dixon WJ, Massey FJ. Introduction to statistical analysis, 4th ed. New York: McGraw-Hill, 1983. Ludbrook J. Multiple comparison procedures updated. Clin Exp Pharmacol Physiol 1998;25:1032. Vucenik I, Passaniti A, Vitolo MI, Tantivejkul K, Eggleton P, Shamsuddin AM. Anti-angiogenic activity of inositol hexaphosphate (IP6). Carcinogenesis 2004;26:2115. Sharma G, Singh RP, Agarwal R. Growth inhibitory and apoptotic effects of inositol hexaphosphate in transgenic adenocarcinoma of mouse prostate (TRAMP-C1) cells. Int J Oncol 2003; 23:1413. Ishikawa T, Nakatsuru Y, Zarkovic M, Shamsuddin, AM. Inhibition of skin cancer by IP6 in vivo: Initiation-promotion model. Anticancer Res 1999;19:3749. Gupta KP, Singh J, Bharathi R. Suppression of DMBA-induced mouse skin tumor development by inositol hexaphosphate and its mode of action. Nutr Cancer 2003;46:66.
Inositol Hexaphosphate (IP6) Inhibits Cellular Proliferation In Melanoma 1 Irfan Rizvi, M.D., Dale R. Riggs, Barbara J. Jackson, Alex Ng, Cynthia Cunningham, Ph.D., and David W. Mcfadden, M.D., F.A.C.S.2 Department of Surgery, Robert C. Byrd Health Science Center, West Virginia University, Morgantown, West Virginia Submitted for publication January 9, 2006
Key Words: melanoma; inositol hexaphosphate; apoptosis; VEGF.
Background. Inositol Hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate found in food sources high in fiber content. We have previously reported IP6 to have significant inhibitory effects against pancreatic cancer in vitro. We hypothesized that the IP6 would significantly inhibit cell growth of cutaneous melanoma in vitro. Materials and methods. The melanoma line HTB68 was cultured using standard techniques and treated with IP6 at doses ranging from 0.2 to 1.0 mM/well. Cell viability was measured by MTT at 72 h. VEGF production was measured in the cell supernatants by ELISA. Apoptosis was evaluated by Annexin V-FITC and results calculated using FACS analysis. Statistical analysis was performed by ANOVA. Results. Significant reductions (P < 0.001) in cellular proliferation were observed with IP6. Overall, IP6 exhibited a mean inhibition of cell growth of 52.1 ⴞ 11.5% (range, 1.6 – 83.0%) at 72 h of incubation. VEGF production was significantly reduced (P < 0.001) by the addition of IP6 (7.5 pg/ml) compared to control (40.9 pg/ml). IP6 significantly increased (P ⴝ 0.029) late apoptosis from 5.3 to 7.0% gated events. No changes in necrosis or early apoptosis were observed. Conclusions. Adjuvant treatment of melanoma continues to challenge clinicians and patients. Our findings that IP6 significantly decreased cellular growth, VEGF production and increased late apoptosis in melanoma suggest its potential therapeutic value. Further in vivo studies are planned to evaluate safety and clinical utility of this agent. © 2006 Elsevier Inc. All rights reserved.
INTRODUCTION
The incidence of melanoma continues to grow, with an estimated 59,580 new cases in 2005. It is estimated that there would be 7,770 mortalities directly because of melanoma in 2005 [1]. After the diagnosis of melanoma, the first course of treatment for locally advanced and/or systemic disease is surgical resection, followed by immunotherapy, chemotherapy and/or radiation therapy [2, 3]. While these treatments are effective in many patients, not all patients will respond and many will have recurrence and or progression of the melanoma. For these patients, new and potentially more effective forms of therapy are continually being investigated. Inositol Hexaphosphate (IP6) is a complex carbohydrate that is a naturally occurring compound found in grains, cereals, nuts, and foods that are high in fiber content. IP6 has been reported to have significant beneficial properties in a number of investigational cancer models, including cancers of the prostate [4, 5], breast [6], colon [7], and pancreas [8]. There has been no reported investigation or evaluation of the effects of IP6 on melanoma. In a continuing effort to evaluate safer and more effective forms of therapy, we investigated the in vitro effects of IP6 on melanoma. MATERIALS AND METHODS
1
Presented at the 1st Annual Academic Surgical Congress, February 7–11, 2006, at the Hyatt Regency Manchester Grand in San Diego, CA. 2 To whom correspondence and reprint requests should be addressed at Department of Surgery, WVU, #1 Medical Center Drive, PO Box 9238, Morgantown, WV 26506. E-mail: dmcfadden@hsc. wvu.edu.
Inositol Hexaphosphate (IP6) IP6 (dodecasodium salt) derived from corn (Sigma Catalogue #P8810) was purchased from Sigma Chemical Co. (St. Louis, MO). IP6 was solubilized in tissue culture media to the desired concentrations of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mM/well.
3
0022-4804/06 $32.00 © 2006 Elsevier Inc. All rights reserved.
4
JOURNAL OF SURGICAL RESEARCH: VOL. 133, NO. 1, JUNE 1, 2006 bind to the antibody. The microplate was then washed to release any unbound substances and an enzyme-lined polyclonal antibody specific for VEGF was added to each well. After incubation, a wash buffer was used to remove any unbound antibody-enzyme reagent and a substrate solution was then added for color development. Once the color development was complete, the plate was read at 540 nm.
Statistical Analysis Determination of statistical significance was performed by analysis of variance [10]. Post hoc comparison of individual concentration means with the control was completed using the Tukey-Kramer Multiple Comparison test [11]. All data are reported as means and standard errors of the means.
RESULTS Reduction of Cellular Proliferation
FIG. 1. Titration of the effects IP6 (corn) on melanoma in vitro evaluated by the MTT assay 72 h incubation.
Cell Culture and Reagents The melanoma cell line HTB68 was purchased from American Type Tissue Collection (ATCC, Manassas, VA). The cell line was cultured and maintained as a monolayer in the preferred media and maintained in monolayer culture at 37°C in humidified air with 5% CO 2. Cells were plated in sterile 96-well plates at 1 ⫻ 105 cells/ml and incubated for 72 h in the presence of IP6 at concentrations listed above, while an equal volume of vehicle was added representing the controls.
MTT Assay The MTT colorimetric assay was performed to detect tumor cell viability after 72 h of incubation. MTT, a tetrazolium dye (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue, Sigma, St. Louis, MO) was added to the each well as described previously [9]. Plates were incubated in the presence of MTT dye for 4 h. Mitochondrial dehydrogenase activity reduced the yellow MTT dye to a purple formazan, which was then solubilized with acidified isopropanol and absorbance was read at 570 nm on an ELISA plate reader.
Significant reductions in cellular proliferation were observed in the HTB68 melanoma cell line by the addition of 0.3 to 1.0 mM/well IP6 (P ⬍ 0.05). The reduction in cellular proliferation ranged from 12.3% ⫾ 4.1 with 0.4 mM/well to 83.0% ⫾ 0.4 with 0.8 mM/well of IP6. Overall, a reduction in cellular proliferation of 52.1% ⫾ 11.5 was observed over doses tested (Fig. 1, P ⬍ 0.001). A dose of 0.35 mM/well was used in subsequent experiments evaluating the effects of IP6 on apoptosis, necrosis, and VEGF production. This dose was chosen based upon its reduction of cellular proliferation observed in the MTT assay (approximately 20%). Evaluation Apoptosis and Necrosis
Late apoptosis was significantly increased by the addition of 0.35 mM/well of IP6 (7.0 ⫾ 0.17) compared to the control (5.3 ⫾ 0.23, P ⫽ 0.029; Fig. 2). No change was noted in either early apoptosis (4.7 ⫾ 0.23) or necro-
Measurement of Apoptosis Annexin V-FITC Apoptosis Detection Kit (Biovision Inc., Mountain View, CA) was used to detect early and late apoptotic activity after 18 h of incubation. After treatment wit 0.35 mM/well of IP6, cells (1 ⫻ 106) were collected and resuspended in binding buffer, and Annexin V-FITC and Propidium Iodide (PI) were added to each sample and incubated in the dark for 5 min. Annexin V-FITC binding was determined by flow cytometry (Ex ⫽ 488 nm; Em ⫽ 530 nm) using FITC signal detector (FL1) and PI staining by the phycoerythrin emission signal detector (FL2).
Human VEGF Quantikine ELISA Kit A commercial ELISA kit (R&D Systems, Minneapolis, MN) was used to detect VEGF (Vascular Endothelial Growth Factor) Production (pg/ml) after 72 h of incubation. After the cells were treated with IP6, the supernatants were collected for quanitification of VEGF production. Cell culture supernatants, standards, or control samples were added in 200 l to a pre-coated microplate containing a monoclonal antibody specific for VEGF. VEGF present in the samples will
FIG. 2. Annexin V determination of apoptotic activity in the HTB68 melanoma in vitro.
RIZVI ET AL.: EFFECTS OF INOSITOL HEXAPHOSPHATE ON MELANOMA
FIG. 3.
VEGF determination of HTB68 melanoma in vitro.
sis (0.1 ⫾ 0.01) by the addition of IP6 in vitro when compared to control (7.2 ⫾ 1.04 and 0.3 ⫾ 0.02, N.S.). Evaluation of In Vitro VEGF Production
IP6 0.35 mM/well significantly reduced the VEGF levels in tissue culture cell supernatants of the HTB68 cells when compared to controls (7.5 ⫾ 1.5 9pg/ml versus 40.9 ⫾ 9. pg/ml, P ⬍ 0.001, Fig. 3). DISCUSSION
Melanoma is a cancerous tumor of melanocytes, with the majority being diagnosed in adults. Early warning signs of melanoma include darkening or change in color, irritation, or itching, and or increased size of existing nevi. Should any of these signs be discovered, a biopsy of the area is indicated. The first line of treatment after detection of melanoma, surgical resection, is a highly effective form of treatment for those patients diagnosed with early stage melanoma. The majority of these patients will not require any additional form of treatment other than surgical resection. However, for patients with advanced stage melanoma (stage III and IV) that has metastasized to the lymph nodes or distant organs, surgical resection alone is not an option. These patients require adjuvant treatment with chemotherapy, immunotherapy, tumor vaccines, or other experimental treatment protocols. However, these forms of treatment are expensive, have substantial co-morbidities and produce marginal benefit in a great number of patients. For this reason the search for more effective and less toxic treatment options for patients suffering from melanoma continues. IP6 is a complex carbohydrate that is a found in grains, cereals, nuts, and foods high in fiber content. IP6 has been reported to significantly decrease the growth of cancers of the prostate [4, 5] breast [6], and
5
colon [7]. We have previously evaluated the in vitro effects of IP6 on human pancreatic cancer [8]. Significant reductions in cell proliferation was observed after 72 h of treatment with IP6 in all doses tested (P ⬍ 0.001). Vucenik et al. [12], also studied the effects of IP6 (dodecasodium salt derived from rice) on cell growth in the bovine aortic endothelial cells (BAECs) and VEGF production in the human liver cancer cell line (HepG2). They observed that IP6 inhibited cellular growth in a dose-dependent manner on day 3 (IC 50 ⫽ 0.74 mM), but lower doses of IP6 (0.1 and 0.3 mM) had no effect on cell growth when compared to the control. However, significant growth inhibition was observed with 0.6 mM of IP6 (84% on day 3 and 62% on day 5). In our current study, significant growth inhibition was observed in the HTB68 melanoma cell line with doses ranging from 0.3 to 1.0 mM/well of IP6 (range of 12.3% to 83.0%) at 72 h of treatment. This data indicates that IP6 may have the potential to become an effective adjuvant treatment for melanoma. Among the reported mechanisms by which IP6 exerts its anti-proliferative effect are through regulation of apoptosis and angiogenesis. Argarwal et al. [5] investigated whether IP6 would inhibit NF-kappa B, which is active in advanced and androgen-independent human prostate cancer cells (DU145), as well as its effects on proliferation and apoptosis. Their group found that when the DU145 cells were treated with doses of 1 and 2 mM of IP6, cellular proliferation was strongly inhibited and apoptotic activity was induced. In other work, IP6 has been shown to significantly increase caspase-3 activity in an experimental mouse prostate model [13]. Sharma et al. evaluated the effects of IP6 in the transgenic adenocarcinoma of mouse prostate (TRAMP-C1) cell line. The TRAMP-C1 cells were treated with various doses of IP6 (ranging from 0.5 to 4.0 mM) for 24 – 72 h. Their results included 6 to 35% cell death, in a dose and time-dependent manner. They also showed a significant induction (six-fold) in caspase-3 activity when compared to control. In the current study, apoptotic activity was measured after 18 h of incubation with 0.35 mM IP6 and no changes were observed in early apoptosis or necrosis; however, late apoptosis was significantly increased when compared to control (P ⫽ 0.029). This data are also supported by our previous work in that apoptotic activity was significantly increased by IP6 in both MiaPaca and PANC-1 human pancreatic cancer cells [8]. VEGF production was also measured to evaluate the anti-angiogenic properties of IP6. VEGF production was significantly reduced with 0.35 mM of IP6 by 81.7% (7.5 ⫾ 0.8 pg/ml) when compared to the control (40.9 ⫾ 4.8 pg/ml) (P ⬍ 0.001). Vucenik et al., also examined the effects of IP6 on VEGF production in HepG2 cells in their two series of experiments. In the first experi-
6
JOURNAL OF SURGICAL RESEARCH: VOL. 133, NO. 1, JUNE 1, 2006
ment, they measured VEGF protein in HepG2 cells treated with increasing concentrations of IP6 (0.5 mM, 1.0 mM, and 2.0 mM), and aliquots of media were removed at 1, 3, 6, and 24 h after incubation. In the second experiment, VEGF protein was measured in HepG2 cells pre-treated with several doses of IP6 (0.5 mM, 1.0 mM, and 2.0 mM) for 24 h. After 24 h, the media was replaced with serum free medium and aliquots were then collected at 3, 6, and 24 h. The researchers found that in the first experiment, IP6 decreased VEGF by approximately 50% after 24 h, in a concentrationdependent manner. However, when the cells were pretreated with various doses of IP6, VEGF production was reduced by 79% at a dose of 1.0 mM, while an 86% reduction of VEGF was observed with 2.0 mM [12]. There have been no reports on the effectiveness of IP6 in melanoma in vivo or in clinical use. However, IP6 has been shown to reduce significantly the development of skin cancer in experimental animal models. Ishikawa et al. studied the effects of 2% IP6 in drinking water on 7,12-dimethylbenz(a)anthracene (DMBA)-induced skin cancer in mice. Animals received IP6 in one of two schedules. The first was the initiation period (3 weeks before tumor induction) and the second was the promotion phase (19 weeks post-tumor induction). The animals receiving IP6 during the initiation period exhibited a 50% reduction in the number of tumors per animal and in the overall incidence of tumor. In contrast, no such inhibition was observed in the group of animals receiving IP6 in the promotion phase [14]. Gupta et al. also investigated the effect of IP6 as a topical agent on DMBA-induced mouse skin tumors. Their results showed that IP6, administered topically, significantly inhibited skin tumor development, presumably by affecting proliferation, differentiation, and by inducing apoptosis [15].
clinical trials to determine the clinical efficacy of IP6 in the management of this disease. REFERENCES 1. 2.
3. 4.
5.
6.
7.
8.
9.
10. 11. 12.
13.
CONCLUSIONS
In summary, we have shown for the first time that IP6 significantly inhibits the in vitro growth of melanoma. Anti-angiogenic and pro-apoptotic mechanisms were demonstrated. The results reported here will lead to in vivo studies, and possibly subsequent phase II
14.
15.
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