Opposing effects of arsenic trioxide on hepatocellular carcinomas in mice

Bing Liu; Shangha Pan; Xuesong Dong; Haiquan Qiao; Hongchi Jiang; Geoffrey W Krissansen; Xueying Sun

doi:10.1111/j.1349-7006.2006.00230.x

. 2006 Apr 28;97(7):675–681. doi: 10.1111/j.1349-7006.2006.00230.x

Opposing effects of arsenic trioxide on hepatocellular carcinomas in mice

Bing Liu ¹, Shangha Pan ¹, Xuesong Dong ¹, Haiquan Qiao ¹, Hongchi Jiang ¹, Geoffrey W Krissansen ², Xueying Sun ^1,^2,^✉

PMCID: PMC11159334 PMID: 16827809

Abstract

Arsenic trioxide (As₂O₃) is a potent antitumor agent used to treat acute promyelocytic leukemia (APL) and, more recently, solid tumors. However, the dose of As₂O₃ required to suppress human xenographs in mice is markedly higher than that used to treat APL in humans. Paradoxically, low doses of As₂O₃ stimulate angiogenesis, which might be expected to promote tumor growth. Clearly, appropriate dosages of As₂O₃ are required to treat human patients to avoid toxicity and undesirable side effects. In the present study, we investigated As₂O₃ with respect to its toxicity and effects on tumor growth, angiogenesis and cell apoptosis using H22 hepatocellular carcinoma (HCC) cells in a mouse model of HCC. As₂O₃ inhibited tumor growth and angiogenesis, and enhanced tumor cell apoptosis at doses greater than 1 mg/kg, but mice lost weight and failed to thrive at doses of 4 mg/kg and greater. In contrast, low doses (<1 mg/kg) of As₂O₃ promoted tumor growth, upregulated the expression of vascular endothelial growth factor and tumor angiogenesis, and had no effect on tumor cell apoptosis. In vitro studies demonstrated that As₂O₃ inhibited the proliferation of H22 tumor cells and bovine aortic endothelial cells, and induced their apoptosis in a dose‐ and time‐dependent fashion, suggesting that the mechanism of As₂O₃‐mediated inhibition of tumor growth is due to direct effects of the drug on both tumor cells and endothelia. In summary, different doses of As₂O₃ have opposing effects on tumor growth and angiogenesis. The results demonstrate that As₂O₃ has a narrow window of therapeutic opportunity with respect to dosage, and that low doses of the drug as used in metronomic therapy should be used with extreme caution. (Cancer Sci 2006; 97: 675–681)

Abbreviations:

AI: apoptosis index
As₂O₃: arsenic trioxide
APL: acute promyelocytic leukemia
BAEC: bovine aortic endothelial cells
HCC: hepatocellular carcinoma
TUNEL: terminal deoxynucleotidyl transferase biotin‐dUTP nick‐end labeling
MTT: 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide
PI: proliferation index
VEGF: vascular endothelial growth factor.

Arsenic trioxide has been used as an anticancer agent in traditional Chinese medicine for several thousand years. It was originally shown to induce complete clinical remission in patients with APL at Harbin Medical University in China in the 1970s.⁽ ¹ ⁾ It has subsequently been widely employed to treat APL, including the treatment of patients that are resistant to conventional chemotherapies.⁽ ² ⁾

The therapeutic potential and antitumor activity of As₂O₃ extends to non‐APL leukemia, and to a variety of solid tumors and tumor cell lines including neuroblastoma,⁽ ³ ⁾ head and neck,⁽ ⁴ ⁾ gastric,⁽ ⁵ ⁾ transitional,⁽ ⁶ ⁾ renal,⁽ ⁷ ⁾ esophageal,⁽ ⁸ ⁾ prostate,⁽ ⁹ ⁾ colorectal⁽ ¹⁰ ⁾ and hepatocellular⁽ ¹¹ ⁾ cancers or cell lines. However, the activity of As₂O₃ against solid tumors has not been as effective as against APL. As₂O₃ induces the apoptosis of various cancer cell lines⁽ ³ , ⁴ , ⁵ , ⁶ , ⁷ ⁾ and inhibits the growth of tumors,⁽ ⁸ , ⁹ , ¹² ⁾ but the dosages of As₂O₃ required to exert these effects⁽ ⁹ , ¹² , ¹³ , ¹⁴ ⁾ are much higher than those required to inhibit hematologic malignancies.⁽ ¹⁵ , ¹⁶ ⁾ Such high doses are not clinically achievable without the risk of severe side effects due to toxicity. A review of recently published reports in which As₂O₃ was used to treat solid tumors in mice reveals that effective dosages of As₂O₃ range from 2 to 6.5 mg/kg in treatments that lasted for 1–6 weeks.⁽ ⁹ , ¹² , ¹³ , ¹⁴ ⁾ Such dosages are considerably higher (approximately 12–40‐fold) than the standard dosage of 0.16 mg/kg used to treat human patients with APL over a period of 6 weeks.⁽ ¹⁵ , ¹⁶ ⁾ Few studies in mice have reported data about the toxicity of As₂O₃.

The anticancer activity of As₂O₃ is in part related to its effects on mitochondria,⁽ ¹⁷ ⁾ and the generation of nitric oxide and reactive oxygen species.⁽ ¹⁸ ⁾ Accordingly, As₂O₃ exerts antitumor effects as a result of its highly cytotoxic nature. Perhaps not surprisingly therefore, systemically administered As₂O₃ has undesirable side effects. As₂O₃ has been reported to exert cardiac toxicity in human patients even at the low dose of 0.16 mg/kg,⁽ ¹⁹ , ²⁰ ⁾ and repeated injection of As₂O₃ produces cardiac toxicities in mice.⁽ ²¹ ⁾ Intraperitoneally administered As₂O₃ has binding affinity for liver and kidneys, and causes damage to both organs.⁽ ²² ⁾ In one recent report, oral administration of As₂O₃ to mice at doses of 3 or 6 mg/kg for 30 days resulted in significant reductions in bodyweight, protein and glycogen, as well as reduction in the ascorbic acid content of both livers and kidneys.⁽ ²³ ⁾ The data suggest that As₂O₃ is highly toxic when systemically administered to mice at the doses required to treat solid tumors. In contrast, low doses of As₂O₃ were shown to stimulate angiogenesis and tumorigenesis.⁽ ²⁴ ⁾ The latter results suggest that the therapeutic dosage of As₂O₃ should be carefully investigated before it is applied to treat solid tumors in patients.

In the present study, we investigated As₂O₃ with respect to its toxicity, and effects on tumor growth, angiogenesis and cell apoptosis in a mouse model of HCC.

Materials and Methods

Mice, cell lines and As₂O₃

Male BALB/c mice (H‐2b), 8 weeks old, were obtained from the Animal Research Center, Harbin Medical University, China. The syngeneic H22 HCC cells and BAEC were supplied by Dianzang Cell Bank of Wuhan University, Wuhan, China. An As₂O₃ solution was purchased from Yida Pharmaceutical Co. Ltd, Harbin Medical University.

Animal experimental design

All procedures administered to animals were in accordance with institutional animal ethics approvals and guidelines. All experiments included 10 mice per treatment group.

Assessment of toxicity Mice were treated by intraperitoneal injection of As₂O₃ at different doses every 2 days. Control mice were injected with an equivalent volume of PBS. The mice were carefully monitored for symptoms of toxicity, and weighed once each week. The mean weight of each group of mice was calculated and used as a parameter of toxicity, as described previously.⁽ ²³ ⁾

Tumor implantation and treatment H22 tumor cells were maintained by successive transplantation into the abdominal cavity of mice. The cancerous ascites were collected, stained with Trypan blue to record viability, and counted by microscopy. Tumors were established by subcutaneous injection of 2 × 10⁵ H22 cells into a site on the left flank of mice, as described previously.⁽ ²⁵ ⁾ Animals were randomly assigned to treatment when tumors reached around 0.2 cm in diameter approximately 7 days after injection of tumor cells. As₂O₃ was administered intraperitoneally at different doses every alternate day. Control mice were given an equivalent volume of PBS. The growth of tumors was monitored and tumor size determined by measuring two perpendicular diameters.

Assessment of tumor vascularity

The methodology to determine tumor vascularity has been described previously.⁽ ²⁵ ⁾ Briefly, 10 µm frozen tumor sections prepared from tumors 4 weeks after treatment were fixed with acetone, rinsed with PBS, blocked with 2% bovine serum albumin for 2 h, and incubated overnight with an anti‐CD31 antibody (MEC13.3, Pharmingen, San Diego, CA, USA). They were subsequently incubated for 30 min with secondary antibodies using the SABC kit (Boster Biological Technology, Wuhan, China), developed with Sigma FAST DAB (3,3′‐diaminobenzidine tetrahydrochloride) and CoCl₂ enhancer tablets (Sigma, St Louis, MO, USA) and counterstained with hematoxylin. Stained blood vessels were counted in five blindly chosen random fields (0.155 mm²) at 400× magnification, and the mean of the highest three counts was calculated.

In situ detection of apoptotic cells

Serial sections of 6 µm thickness were prepared from tumors 4 weeks after treatment, stained with the TUNEL agent (Boehringer Mannheim, Mannheim, Germany), and examined by fluorescence microscopy. Adjacent sections were counterstained with hematoxylin and eosin. The total number of apoptotic cells in 10 randomly selected fields was counted. The AI was calculated as the percentage of positive‐staining cells, namely:

AI = number of apoptotic cells × 100/total number of nucleated cells.

Proliferation, viability and apoptosis of cells in vitro

The cells (5 × 10³ H22 or 1 × 10⁴ BAEC) were seeded in 96‐well plates in 200 µL of RPMI‐1640 media (Life Technologies, Beijing, China) supplemented with 50 U/mL penicillin/streptomycin. They were cultured at 37°C for 24 h, and the medium was replaced with fresh RPMI‐1640 or the same media containing different concentrations of As₂O₃. After a further 12, 24, 36 or 48 h incubation, 20 µL of MTT was added to each well followed by incubation for 4 h. The medium was discarded and 150 µL of dimethylsulfoxide was added into each well and incubated for 20 min. The OD_570nm was measured and the PI calculated according to the formula:

experimental OD value/control OD value × 100%.

To measure cell apoptosis, the cells harvested after As₂O₃ treatment as above were fixed with 4% paraformaldehyde solution, and permeabilized with a solution of 0.1% Triton‐X100 and 0.1% sodium citrate. They were incubated with TUNEL reagent for 60 min at 37°C, examined by fluorescence microscopy, and the AI was calculated as above. The experiments were repeated three times.

Expression of VEGF by western blot analysis

Tumors excised from mice 4 weeks after treatment were minced, and homogenized in protein lysate buffer. Debris was removed by centrifugation, and the lysates were resolved on 10% polyacrylamide sodium dodecylsulfate gels, and electrophoretically transferred to nitrocellulose Hybond C extra membranes (Amersham Life Sciences, Buckinghamshire, UK). The membranes were incubated with an anti‐VEGF monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by a horseradish peroxidase‐conjugated secondary antibody, and developed by enhanced chemiluminescence (Amersham) followed by exposure to X‐ray film. The density of each band was evaluated using a densitometric analysis program (FR200; Furi, Shanghai, China). Blots were stained with an antitubulin antibody to confirm that each lane contained similar amounts of tumor homogenate. The relative levels of VEGF were normalized with respect to tubulin band density.

H22 cells were cultured in 2 mL of RPMI‐1640 or the same media containing different concentrations of As₂O₃ for 36 h, and the levels of VEGF in cell lysates were measured as above.

Statistical analysis

Results were expressed as mean values ± SD, and a Student's t‐test was used for evaluating statistical significance. A value of less than 0.05 (P < 0.05) was used for statistical significance.

Results

Toxicity of As₂O₃ in mice

Different doses of As₂O₃ (0.5, 1, 2, 4 and 8 mg/kg) were administered intraperitoneally every 2 days for 6 weeks into mice weighing on average 24.8 ± 1.2 g. Systemic administration of As₂O₃ at a dose of either 4 or 8 mg/kg caused the coats of mice to become dull and lose hair, pigmented spots appeared on the skin, and hyperkeratinosis was evident. The feet of mice showed signs of swelling, and mice became lethargic. No mice died as a consequence of As₂O₃ administration. Weight loss was used as a parameter to quantify toxicity. The results demonstrated that As₂O₃ had no significant effect on the weight of mice when administered at a dose of 0.5 or 1 mg/kg (Fig. 1). There was no significant difference in bodyweight between mice treated with PBS and As₂O₃ at a dose of 2 mg/kg until the sixth week when a significant difference (8% reduction, P < 0.05) in bodyweight was observed, suggesting that the effects of As₂O₃ are cumulative (Fig. 1). However, when As₂O₃ was administered at doses of 4 and 8 mg/kg, the difference in weight became significant (11 and 16% reduction, both P < 0.05) and highly significant (19 and 28% reduction, both P < 0.01) after the fourth and sixth weeks, respectively. The results suggest that for long‐term treatment, As₂O₃ should not be administered to mice at a dosage over 2 mg/kg in order to avoid severe side effects.

Effects of As₂O₃ on the body weight of mice. Different doses of As₂O₃ (0.5, 1, 2, 4, and 8 mg/kg) were administered i.p. to 8‐week‐old male BABL/c mice every 2 days. Control mice were injected with phosphate‐buffered saline (PBS). *P < 0.05, significantly different from control mice.

Effects of As₂O₃ on tumor growth

Tumors were established by subcutaneous injection of 2 × 10⁵ H22 cells into the flanks of mice. Animals were randomly assigned to treatment when tumors reached around 0.2 cm in diameter after approximately 7 days. As₂O₃ was administered inrtaperitoneally at doses of 0.2, 0.5, 1, 2 and 4 mg/kg every 2 days for 30 days. Tumor growth became significantly inhibited (by 17, 35 and 53%, respectively, all P < 0.05) by the 20th day of treatment with doses of 1, 2 and 4 mg/kg of As₂O₃, and by the 15th day in the case of the highest dose of 4 mg/kg (46% reduction, P < 0.05). In marked contrast, the two lowest doses of 0.2 and 0.5 mg/kg had significantly promoted the growth of tumors (19 and 15% increase, respectively, both P < 0.05) by the 30th day of treatment compared to the growth of tumors of control mice (Fig. 2).

Effects of As₂O₃ on tumor angiogenesis

The vascularity of tumors of mice 4 weeks after treatment was examined by enumeration of vessels in tumor sections stained with an anti‐CD31 monoclonal antibody. Low doses of As₂O₃ surprisingly promoted tumor angiogenesis as evidenced by increased blood vessel density following treatment (Fig. 3a,b). There was a significant increase (37%, P < 0.01) in tumor blood vessel counts of mice treated with 0.2 mg/kg As₂O₃ compared to mice treated with PBS. In contrast, As₂O₃ at a dose of 0.5 mg/kg only slightly increased vessel counts such that there was no significant difference compared to control mice. High doses of As₂O₃ had an opposing effect in that tumor blood vessel density was significantly decreased (by 25–42%, P < 0.05) in a dose‐dependent fashion when the dosage of As₂O₃ was raised to either 1, 2 or 4 mg/kg (Fig. 3a,b).

Effects of As₂O₃ on tumor vascularity and expression of vascular endothelial growth factor (VEGF). (a) Representative sections from tumors of mice treated by i.p. administration of phosphate‐buffered saline (PBS), 0.2 mg/kg of As₂O₃, or 4 mg/kg of As₂O₃ for 4 weeks. Sections were stained with an anti‐CD31 mAb to visualize blood vessels. (b) The blood vessels were counted to record mean vessel density. *A significant (P < 0.05) increase in mean vessel counts of tumors treated with As₂O₃ versus PBS (control). §A significant reduction in mean vessel counts of tumors treated with As₂O₃ versus PBS (control). (c) Western blot analysis of VEGF expression in homogenates of tumors from mice treated with PBS (control, lane 1) or As₂O₃ (lane 2: 0.2 mg/kg; lane 3: 0.5 mg/kg; lane 4: 1 mg/kg; lane 5: 2 mg/kg; lane 6: 4 mg/kg). (d) Western blot analysis of VEGF expression in lysates of H22 cells incubated with PBS (control, lane 1) or As₂O₃ (lane 2: 0.1 µg/mL; lane 3: 0.2 µg/mL; lane 4: 0.4 µg/mL; lane 5: 0.8 µg/mL). The density of each band was measured and compared to that of the internal control, tubulin. *A significant difference at P < 0.05 in the band intensities of VEGF between control and As₂O₃‐treated groups.

Effects of As₂O₃ on expression of VEGF in vivo and in vitro

Given that As₂O₃ affected the vascularity of tumors, we investigated whether it affected the expression of VEGF, a key promoter of angiogenesis commonly expressed in tumors. Western blot analysis of tumor homogenates revealed, surprisingly, that As₂O₃ upregulated the expression of VEGF (by 89–140%, P < 0.01) at all of the doses of As₂O₃ used in this study compared with PBS‐treated control tumors. Quantification of the densities of bands on the blot indicated that there was no significant difference in the levels of VEGF induced in response to different doses of As₂O₃, implying that low and high doses of As₂O₃ induce VEGF expression to similar extents (Fig. 3c). Similarly, western blot analysis of cell lysates also showed that As₂O₃ upregulated the expression of VEGF at all of the concentrations of As₂O₃ used in this study compared with the control. Quantification of the densities of bands on the blot indicated that there was no significant difference in the levels of VEGF induced in response to different concentrations of As₂O₃, in accordance with the in vivo studies (Fig. 3d).

As₂O₃ induces cell apoptosis in situ

We examined whether tumors underwent programmed cell death after administration of As₂O₃, as measured by in situ labeling of fragmented DNA using the TUNEL method. Adjacent sections were stained with hematoxylin and eosin, and the AI was calculated. The AI for tumors treated with As₂O₃ at doses of 1, 2 and 4 mg/kg was significantly higher (by 39, 51 and 69%, respectively, all P < 0.01) than that for PBS‐treated tumors. However, As₂O₃ treatment at the lowest dose of 0.2 mg/kg did not increase the AI of tumors. The dose of 0.5 mg/kg of As₂O₃ slightly increased the AI compared to that of control tumors, but the difference was not significant (Fig. 4a,b). The results indicate that As₂O₃ induces tumor cell apoptosis in a dose‐dependent manner.

As₂O₃ induces tumor cell apoptosis *in situ*. (a) Illustrated are representative terminal deoxynucleotidyl transferase biotin‐dUTP nick‐end labeling (TUNEL)‐stained sections from the tumors of mice treated by i.p. administration of either phosphate‐buffered saline (PBS), 0.2 mg/kg of As₂O₃, or 4 mg/kg of As₂O₃ for 4 weeks. (b) TUNEL‐positive cells in tumor sections from mice treated by i.p. administration of either PBS, or 0.2, 0.5, 1, 2 or 4 mg/kg of As₂O₃ for 4 weeks, were counted to record the apoptosis index. *A significant difference at P < 0.01 in tumor cell apoptosis between the tumors of PBS (control) and As₂O₃‐treated mice.

As₂O₃ inhibits the proliferation and viability of BAEC and H22 cells and induces their apoptosis in vitro

Angiogenesis is a complex process that encompasses activation, migration and proliferation of endothelial cells. We investigated the effects of As₂O₃ in vitro on BAEC to determine whether As₂O₃ has direct effects on vascular endothelial cells. As₂O₃ inhibited the proliferation and viability of BAEC and induced their apoptosis in a dose‐ and time‐dependent manner. Differences in both the PI and AI of the As₂O₃‐ and PBS‐treated groups became significant at concentrations of 0.2 µg/mL or greater, whereas concentrations less than 0.2 µg/mL had no significant effect (Fig. 5a,b). As₂O₃ also inhibited the proliferation and viability of H22 cells and induced their apoptosis in a dose‐ and time‐dependent manner (Fig. 5c,d). Furthermore, at the same concentration, As₂O₃ was shown to have stronger inhibitory effects on H22 tumor cells than on BAEC.

As₂O₃ inhibits the proliferation of H22 cells and BAEC and induces their apoptosis *in vitro*. H22 cells and BAEC were incubated in the absence or presence of As₂O₃ at different concentrations, and harvested at different time‐points for analysis. The proliferation/viability of H22 cells (a) and BAEC (b) was assessed using the MTT method and the proliferation index was calculated. The apoptosis of H22 cells (c) and BAEC (d) was assessed using terminal deoxynucleotidyl transferase biotin‐dUTP nick‐end labeling (TUNEL) analysis and the apoptosis index was calculated. *A significant difference at P < 0.05; **a highly significant difference at P < 0.001, from control at the indicated time‐point.

Discussion

The present study has for the first time simultaneously investigated the toxicity of systemically delivered As₂O₃, and its effects on tumor growth, angiogenesis and apoptosis in a mouse model of HCC. The data indicate that different doses of As₂O₃ have opposing effects on HCC tumor growth and angiogenesis. Dosages ranging from 1 to 2 mg/kg are appropriate for treating HCC tumors in mice, as tumor growth is suppressed and the side effects are endurable. It is not known whether the results are translatable to the affects of As₂O₃ in human cancer patients, but the fact that the optimum dosage of cancer drugs can vary markedly between different species warrants follow‐up studies in humans.

Tumors must establish an adequate vascular network to facilitate metastasis and acquire the nutrition necessary for growth. The dependency of tumors on angiogenesis provides the rationale for antiangiogenesis therapy of cancers aimed at shutting down the tumor blood supply. The frequent or continuous administration of low doses of chemotherapeutic agents, denoted metronomic chemotherapy, exerts a marked antiangiogenic effect, resulting in a superior antitumor effect compared with conventional scheduling.⁽ ²⁶ ⁾ These findings herald the start of a new era in chemotherapy, as metronomic treatment targeting genomically stable endothelial cells may reduce the risk of development of drug resistance while reducing the toxic side effects of drugs. A number of chemotherapeutic drugs have been shown to inhibit the tumor angiogenesis of several different types of cancers when administered at low doses (metronomic).⁽ ²⁶ ⁾ However, the metronomic administration of As₂O₃ might not be desirable to treat solid tumors as it stimulates tumor angiogenesis when administered at low doses.⁽ ²⁴ , ²⁷ ⁾ Low doses of As₂O₃ have been demonstrated to increase endothelial cell growth and tube formation in three dimensions⁽ ²⁸ ⁾ and induce the expression of VEGF in endothelial and cervical cancer cells,⁽ ²⁸ , ²⁹ , ³⁰ ⁾ but are unable to induce apoptosis of endothelial cells.⁽ ³¹ ⁾ In the studies in which As₂O₃ was shown to inhibit angiogenesis, it was used at a very high dose.⁽ ³² , ³³ ⁾ In the present study we demonstrated that As₂O₃ induced the expression of VEGF at both low and high doses, but the apoptosis of vascular endothelial cells was induced only at high concentrations. The validity and generality of augmented synthesis of VEGF by As₂O₃ treatment has already been established by several prior studies investigating the effects of As₂O₃ or arsenite on B16 melanoma,⁽ ²⁴ ⁾ ovarian cancer cells,⁽ ²⁹ , ³⁴ ⁾ and human umbilical vein endothelial cells,⁽ ²⁸ ⁾ yet Roboz et al. reported that As₂O₃ inhibited the expression of VEGF in a leukemic cell line HEL.⁽ ³² ⁾ The opposing effects of As₂O₃ on VEGF expression are probably cell‐specific and dose‐specific. Notably, the dose of As₂O₃ used by Roboz et al. was higher than the highest dose used in the present study. We hypothesize that low doses of As₂O₃ promote tumor angiogenesis because they upregulate the expression of VEGF, but are unable to induce the apoptosis of endothelial cells. In contrast, high doses of As₂O₃ inhibit tumor angiogenesis because the apoptosis of vascular endothelial cells by As₂O₃ overweighs upregulation of VEGF. In accord, low concentrations of As₂O₃ significantly increased vascularity and blood vessel density in a chorioallantoic membrane assay of angiogenesis, whereas high concentrations of As₂O₃ generated pools of blood instead of vessels.⁽ ³⁰ ⁾ As₂O₃ induces apoptosis in several cancer cell lines through either activation of caspase‐3, caspase‐7 and caspase–9,⁽ ⁹ , ³⁵ , ³⁶ ⁾ downregulation of the anti‐apoptotic protein Bcl‐2,⁽ ³⁵ ⁾ production of free radical species,⁽ ³⁶ , ³⁷ ⁾ activation of c‐Jun NH₂‐terminal kinase and p38,⁽ ⁹ , ³¹ ⁾ or induction of p53 protein.⁽ ³¹ ⁾

Emerging evidence suggests that angiogenesis plays a seminal role in the proliferation of liquid tumors, where endothelial cells release cytokines that stimulate the growth of leukemic cells, and leukemic cells in turn release endothelial growth factors such as VEGF.⁽ ³² ⁾ As₂O₃ interrupts this reciprocal stimulatory loop between leukemic cells and endothelial cells by causing apoptosis of both cell types and by inhibiting VEGF production by leukemic cells.⁽ ³² ⁾ The potent antileukemic effect of As₂O₃ is attributed, in part, to the ‘fluid’ feature of leukemia, which allows high local concentrations of As₂O₃ and close contact with cancer cells. The results presented herein indicate that caution should be taken in the systemic administration of As₂O₃ to treat HCC, and possibly other solid tumors. As₂O₃ is effective against solid tumors, but there is a narrow window of therapeutic opportunity, given that low doses of As₂O₃ stimulate tumor growth and high doses are toxic. In an alternative approach, localized delivery of As₂O₃ to treat solid tumors has been used to achieve therapeutic efficacy and reduce toxicity. Delivery of As₂O₃ directly to the tumor improves the tumor/normal tissue uptake ratios, and permits higher and more frequent doses of the anticancer drug, and at the same time avoids toxicity by reducing uptake of the drug by other organs.⁽ ⁸ , ³⁸ , ³⁹ ⁾ Alternatively, lower doses of As₂O₃ could be effectively used if As₂O₃ was to be combined with other therapeutic modalities such as B7H3‐mediated immunotherapy,⁽ ²⁵ ⁾ non‐calcemic vitamin D analogs,⁽ ⁴⁰ ⁾ l‐buthionine‐sulfoximine⁽ ⁴¹ ⁾ or docosahexaenoic acid.⁽ ⁴² ⁾

Acknowledgments

This work was financially supported by the National Natural Scientific Foundation of China (30471681, 30571808), and the Collaborative Fund for Overseas Scholars from the Scientific and Technological Bureau of Heilongjiang Province, China (WH05C02).

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[b32] 32. Roboz GJ, Dias S, Lam G et al. Arsenic trioxide induces dose‐ and time‐dependent apoptosis of endothelium and may exert an antileukemic effect via inhibition of angiogenesis. Blood 2000; 96: 1525–30. [PubMed] [Google Scholar]

[b33] 33. Lew YS, Brown SL, Griffin RJ, Song CW, Kim JH. Arsenic trioxide causes selective necrosis in solid murine tumors by vascular shutdown. Cancer Res 1999; 59: 6033–7. [PubMed] [Google Scholar]

[b34] 34. Duyndam MC, Hulscher STM, Van Der Wall E, Pinedo HM, Boven E. Evidence for a role of p38 kinase in hypoxia‐inducible factor 1‐independent induction of vascular endothelial growth factor expression by sodium arsenite. J Biol Chem 2003; 278: 6885–95. [DOI] [PubMed] [Google Scholar]

[b35] 35. Li X, Ding X, Adrian TE, Path FRC. Arsenic trioxide causes redistribution of cell cycle, caspase activation, and GADD expression in human colonic, breast, and pancreatic cancer cells. Cancer Invest 2004; 22: 389–400. [DOI] [PubMed] [Google Scholar]

[b36] 36. Chen YC, Lin‐Shiau SY, Lin JK. Involvement of reactive oxygen species and caspase 3 activation in arsenite‐induced apoptosis. J Cell Physiol 1998; 177: 324–33. [DOI] [PubMed] [Google Scholar]

[b37] 37. Jing Y, Dai J, Chalmers‐Redman RM, Tatton WG, Waxman S. Arsenic trioxide selectively induces acute promyelocytic leukemia cell apoptosis via a hydrogen peroxide‐dependent pathway. Blood 1999; 94: 2102–11. [PubMed] [Google Scholar]

[b38] 38. Zhu AL, Liu LX, Piao DX, Lin YX, Zhao JP, Jiang HC. Liver regional continuous chemotherapy: use of femoral or subclavian artery for percutaneous implantation of catheter‐port systems. World J Gastroenterol 2004; 10: 1659–62. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b39] 39. Gochi A, Orita K, Fuchimoto S, Tanaka N, Ogawa N. The prognosis advantage of preoperative intratumoral injection of OK‐432 for gastric cancer patients. Br J Cancer 2001; 84: 443–51. [DOI] [PMC free article] [PubMed] [Google Scholar]

[b40] 40. Kumagai T, Shih LY, Hughes SV et al. 19‐Nor‐1,25(OH) 2D2 (a novel, noncalcemic vitamin D analogue), combined with arsenic trioxide, has potent antitumor activity against myeloid leukemia. Cancer Res 2005; 65: 2488–97. [DOI] [PubMed] [Google Scholar]

[b41] 41. Maeda H, Hori S, Ohizumi H et al. Effective treatment of advanced solid tumors by the combination of arsenic trioxide and 1‐buthionine‐sulfoximine. Cell Death Differ 2004; 11: 737–46. [DOI] [PubMed] [Google Scholar]

[b42] 42. Baumgartner M, Sturlan S, Roth E, Wessner B, Bachleitner‐Hofmann T. Enhancement of arsenic trioxide‐mediated apoptosis using docosahexaenoic acid in arsenic trioxide‐resistant solid tumor cells. Int J Cancer 2004; 112: 707–12. [DOI] [PubMed] [Google Scholar]

PERMALINK

Opposing effects of arsenic trioxide on hepatocellular carcinomas in mice

Bing Liu

Shangha Pan

Xuesong Dong

Haiquan Qiao

Hongchi Jiang

Geoffrey W Krissansen

Xueying Sun