HVJ-liposome-mediated transfection of HSVtk gene driven by AFP promoter inhibits hepatic tumor growth of hepatocellular carcinoma in SCID mice

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Abstract

Suicide gene therapy using ganciclovir (GCV) with transfection of the herpes thymidine kinase (HSVtk) gene has been studied for cancer therapy. The present study demonstrates an efficient method of suicide gene therapy for multiple hepatic tumors, involving repetitive transfection of the HSVtk gene driven by the alpha-fetoprotein (AFP) promoter using hemagglutinating virus of Japan (HVJ)-liposomes. AFP-producing cells (HUH7) and AFP-nonproducing cells (LS180) were injected subcutaneously (s.c.) to establish tumors in nude mice. Two plasmid constructs, bacterial LacZ gene driven by the AFP promoter (AFPLacZ), and HSVtk gene driven by the AFP promoter (AFPTK1) were encapsulated into the HVJ-liposome and used. When AFPLacZ was injected into the s.c. tumors, expression of LacZ gene was confined to HUH7 tumors. Repeated transfection of AFPTK1 followed by GCV treatment markedly suppressed growth of HUH7 tumors, and apoptosis of HUH7 cells was recognized in the tumor. Next, HUH7 cells were injected into the portal vein in severe combined immunodeficiency mice to establish a hepatic tumor model. After inoculation with the tumor, HVJ-liposomes containing the AFPTK1 plasmid vector were injected into the portal vein via the splenic hilum, followed by GCV treatment. This gene therapy significantly inhibited the growth of tumors in the liver and markedly improved survival. Three injections of the AFPTK1 plasmid vector completely inhibited tumor growth. This procedure seems to have great potential for the treatment of multiple hepatic tumors.

Main

Hepatocellular carcinoma (HCC) is one of the most common human malignancies in the world, especially in eastern Asia and Africa.1 An increase in the number of cases of HCC has also occurred in the United States of America over the past two decades.2 Although several therapeutic options are available including surgery, the prognosis of this disease is still poor. The existence of multiple tumors and recurrence after treatment are common in patients with HCC,3 suggesting that gene therapy may be a useful strategy for treating multiple liver tumors in these patients.

Adenovirus-mediated or retrovirus-mediated suicide gene therapy using the HSVtk gene under the control of the AFP promoter has been reported.45 Although this therapy was effective in vitro and for subcutaneous (s.c.) tumors, the effect on hepatic tumors was not clarified in that study. In the present study, we tested hemagglutinating virus of Japan (HVJ) liposome-mediated AFP-HSVtk gene transfer directly into subcutaneous tumors. Moreover, we transferred HVJ-liposome containing AFP-HSVtk gene via the portal vein for the treatment of multiple tumors in mouse liver. Inhibition of hepatic tumor growth was marked and the repeated transfection increased the effect.

We have previously shown that an adenovirus vector containing the AFPTK1 plasmid can specifically kill AFP-producing cells in vitro and in s.c. tumors.4 To develop a feasible treatment for multiple hepatic tumors of HCC, we employed HVJ-liposome6 to test the efficacy of gene tranduction in liver tumors. Retroviral vectors have been used for gene delivery into rat livers in combination with a partial hepatectomy,78 but transfection efficiency was relatively low and gene expression was observed in 5–15% of hepatocytes. Adenoviral vectors appear to be more efficient for gene induction in hepatocytes in vivo,9 but these vectors stimulate a host immune response and do not allow repeated transfections. HVJ-liposomes contain the envelope of hemagglutinating virus of Japan (Sendai virus), and show fusion ability.10 DNA encapsulated in liposomes was successfully introduced into cells by making use of the fusion activity of HVJ. Recently, it was reported that HVJ-liposome can achieve exogenous gene transfection into hepatocyte with high efficiency and repeated in vivo transfection was shown to prolong the expression.6 Furthermore, antibody response to HVJ-liposome after transfection was weak and transient, and HVJ-liposome-transfected animals did not generate CTL activity.6 A human hepatocellular carcinoma cell line, HUH7, and a human colon cancer cell line, LS180, were used in this study. HUH7 cells express AFP, whereas LS180 cells do not express AFP.4 We first injected HVJ-liposomes containing AFPLacZ plasmid vector into the s.c. HUH7 or LS180 tumors to assess the specificity of the AFP promoter. The expression of LacZ gene in the s.c. tumor was examined by staining with X-gal. A number of cells were stained with X-gal in AFP-producing HUH7 tumors whereas no cells were stained with X-gal in AFP-nonproducing LS180 tumors (Figure 1a and b). The result implies that specific gene expression in the AFP-producing tumor was obtained by the AFP promoter in vivo. We next injected HVJ-liposomes containing AFPLacZ or AFPTK1 into the s.c. HUH7 tumors to investigate apoptosis of the tumor cells. Apoptotic cells were assessed by TUNEL, which detects fragmented DNA in situ (Figure 1c and d). Apoptotic cells were found in the tumors injected with AFPTK1 followed by GCV treatment, but no apoptotic cells were detected in the tumors injected with AFPLacZ followed by GCV administrations. Moreover, DNA ladder formation was observed in tumors injected with AFPTK1 followed by GCV (Figure 1e). The growth suppression of s.c. tumors of HUH7 by AFPTK1 transfection is shown in Figure 2. Repeated injection of AFPTK1 followed by GCV treatment completely suppressed tumor growth. In contrast, there was no tumor suppression in the other three groups.

Figure 1
figure1

LacZ expression in tumors examined by X-gal staining (a and b). Four mice were given s.c. injections in the right flank with 1 × 107 LS 180 cells or with 1 × 107 HUH7 cells. Staining of the LacZ was observed in HUH7 tumor (a), but not in LS180 tumor (b) (× 40). Another four HUH7 injected mice received AFPLacZ or AFPTK1 injections on 2 consecutive days and GCV treatment for 6 days. Apoptosis of HUH7 cells was found in HUH7 tumors transfected with AFPTK1 followed by GCV treatment (c), but not in HUH7 tumors transfected with AFPLacZ followed by GCV treatment (d) (× 160). Brown cells indicate the apoptotic cells detected by TUNEL method. DNA ladder formation (e). Lanes 1 and 2, DNA from HUH7 tumors transfected with AFPLacZ followed by GCV treatment. AFPLacZ contains bacterial LacZ gene under the control of the AFP promoter. Lanes 3 and 4, DNA from HUH7 tumors transfected with AFPTK1 followed by GCV treatment. AFPTK1 contains the 4.9 kb fragment of the 5′-flanking sequence of the AFP gene and the tripartite leader upstream of the HSV-TK gene.4 The plasmid vector AFPLacZ or AFPTK1 was encapsulated into liposomes containing HVJ-liposomes, as described previously.11 Briefly, phosphatidylserine, phosphatidylcholine, and cholesterol were mixed and dried. This dried lipid and 10 μg of plasmid DNA, which had been complexed with 12 μg of high-mobility group-1 (HMG1) nonhistone chromosomal protein purified from calf thymus, were shaken vigorously and sonicated to form liposomes. UV-irradiated HVJ (30 000 hemagglutinating units) was mixed with the liposome suspension, and residual free HVJ was removed from the HVJ-liposomes by sucrose density gradient centrifugation. To detect apoptosis in the tumor, a modified TUNEL method11 was performed using Apop Tag In Situ Apoptosis Detection System (Oncor, Gaitherburg, MD, USA). DNA was extracted from the subcutaneous tumors using the Isoquick kit (ORCA, Bothell, WA,USA), treated with 100 μg/ml of Rnase (Sigma, St Louis, MO, USA) for 1 h, electrophoresed in a 1.5% agarose gel (FMC, Rockland, ME, USA) and visualized by staining with ethidium bromide.

Figure 2
figure2

Growth suppression of s.c. HUH7 tumors after repeated injections of plasmid vectors. Subcutaneous tumors were established using athymic BALB/c nude mice (6-week-old). Twenty-four mice were given s.c. injections in the right flank with 1 × 107 HUH7 cells. Mice were divided into four groups according to the treatment schedules: AFPTK1 injection and GCV treatment (n = 6); AFPTK1 injection without GCV treatment (n = 6); AFPLacZ injection with GCV treatment (n = 6); GCV treatment only (n = 6). Two weeks after injection of HUH7 cells, 250 μl of HVJ-liposome containing 10 μg of AFPTK1 vector or AFPLacZ vector was directly injected into the growing tumor from three directions with 30 gauge-needles for 2 consecutive days. After 2 consecutive days of AFPTK1 injections, GCV was administrated i.p. at 100 mg/kg once daily for 6 days. The transfection followed by GCV treatment was done repeatedly 10 times. Size of tumor was measured twice weekly with calipers in three dimensions. Tumor size is presented as the mean ± s.e. mm3.

Visible tumor foci are generally formed in the liver within a week of injection of HUH7 cells, and these grew to a few millimeters in diameter by day 21. We injected HVJ-liposome containing AFPLacZ into the portal vein through the splenic hilum 7 days after tumor inoculation to assess the specificity of the AFP promoter in the hepatic tumor. LacZ gene expression was observed in the HUH7 tumor, but not in normal liver tissue by X-gal staining (Figure 3). Treatment was initiated 7 days after tumor cell injection to test whether AFPTK1 gene therapy followed by GCV treatment could inhibit the growth of tumors derived from hepatic micrometastases already present at the time of treatment. Figure 4a shows representative results of gene therapy. Most of the control mice developed multiple large ‘cannon ball’ tumors in the liver, as seen in Figure 4a, panel 1. HVJ-liposome-mediated gene transfer of AFPTK1 followed by GCV treatment markedly inhibited tumor growth, and repeated transfection increased the effect (Figure 4a, panels 2–4).

Figure 3
figure3

LacZ expression in hepatic tumors of HUH7 examined by X-gal staining. Hepatic tumors were established by the method of Kozlowski et al12 with some modifications. Severe combined immunodeficient (SCID) mice (6 weeks old; Clea Japan, Tokyo, Japan) were anesthetized, and a transverse incision was made in the left flank through the skin and peritoneum to expose the spleen. Then, 5 × 106 HUH7 cells in 250 μl of RPMI 1640 containing 10% fetal bovine serum were injected into the portal vein through the splenic hilum using a 30-gauge needle. For natural killer cell depletion in vivo, SCID mice were injected intravenously with 20 μg of anti-asialo GM1 rabbit serum 5, 7, 9 and 12 days after tumor cell injection. HVJ-liposomes containing 10 μg of the AFPLacZ vector were injected into the portal vein via the splenic hilum 7 days after tumor inoculation.

Figure 4
figure4

(a) Representative results of gene therapy in SCID mice. Panel 1, control mice; panel 2, mice received a single injection of AFPTK1; panel 3, mice received two injections of AFPTK1; panel 4, mice received three injections of AFPTK1. (b) Effect of AFPTK1 on hepatic tumors of HUH7. Hepatic tumors were established as described in Figure 3. HVJ-liposomes containing 10 μg of the AFPTK1 vector were injected into the portal vein via the splenic hilum. Mice were divided into four groups according to their treatment: (1) a single injection of AFPTK1 at 7 days after tumor inoculation (n = 12); (2) AFPPTK1 at 7 and 21 days after tumor inoculation (n = 9); (3) AFPPTK1 at 7, 21 and 35 days after tumor inoculation (n = 7); and (4) control mice receiving phosphate-buffered saline at 7 days after tumor inoculation (n = 13). All mice received ganciclovir (GCV) intraperitoneally at 100 mg/kg for 10 days after each transfection. Control mice began to die 48 days after HUH7 inoculation. When an animal died, the liver was removed and fixed in formaldehyde for 3 days. The weight of all hepatic tumors was determined after careful separation from the normal surrounding liver.13

A single injection of AFPTK1 had a substantial antitumor effect at 10 weeks (Figure 4b). Nine of 12 mice had no detectable hepatic tumors at the time of death, two had tumors of <1 mg, and one had tumors weighing 5.4 mg. The antitumor effect of AFPTK1 was significant by the Mann–Whitney rank sum test (P = 0.042 versus untreated; 5.21 ± 3.88 g). Mice were also treated by repeated injection of the AFPTK1 vector. Two injections (at 7 and 21 days after tumor inoculation) and three injections (at 7, 21 and 35 days after tumor inoculation) boosted the antitumor effect (P = 0.009 and P = 0.007, respectively).

We also determined the duration of survival when SCID mice were treated with AFPTK1 encapsulated in HVJ-liposomes (Figure 5). Placebo-treated mice began to die 47 days after tumor cell injection and the majority was dead by 10 weeks (median survival time: 58 days; n = 13). In contrast, mice transfected with a single injection of AFPTK1 survived significantly longer than control mice and 11 of 12 mice were alive at 10 weeks (P < 0.05, compared with control mice by log rank analysis of Kaplan–Meier curves). Two injections of AFPTK1 also achieved better survival than seen in control mice (P < 0.05). Three injections completely prevented death, and the mice were free from hepatic tumors. One mouse that received two injections of AFPTK1 died although the tumor was not highly expanded in the liver (total tumor weight was 2.1 g). No extrahepatic metastatic lesion was observed in the mouse. Although the precise cause of death in the mouse was not clear, it is possible that the tumor developed near the hilus of the liver and thus became fatal.

Figure 5
figure5

Kaplan–Meier curves for mice treated with AFPTK1. All surviving mice were killed 10 weeks after HUH7 inoculation. The survival rate of mice treated with AFPTK1 and GCV was compared with that of control mice.

We injected AFPTK1 vector encapsulated in HVJ-liposomes into mice 8 weeks after tumor cell injection. Two of six mice died under anesthesia and all others died within a week of AFPTK1 injection: all six mice developed large hepatic tumors (data not shown). Thus, HVJ-liposome-mediated AFPTK1 gene therapy should be considered for smaller-sized tumors.

Here, we have demonstrated that repeated transfections with the AFPTK1 vector inhibited hepatic growth of HCC, resulting in the survival of mice with an otherwise lethal illness. Although the present study used a SCID mouse model which lacks immune response, we have shown that repeated in vivo transfection into rat liver using HVJ-liposomes can be done without causing substantial inflammation or activation of the cellular and humoral immune responses.6 HVJ-liposome injection via the hepatic artery or via the portal vein through the splenic artery will be clinically feasible using a catheter with a subcutaneous port. Therefore, the present approach may be useful for the treatment of small multiple HCC or for prevention of metastasis after surgical resection.

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Acknowledgements

This work was supported by a Grant-in Aid for Scientific Research provided by the Ministry of Education, Science and Culture of Japan (No. 07457282).

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Correspondence to J Fujimoto.

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Hirano, T., Kaneko, S., Kaneda, Y. et al. HVJ-liposome-mediated transfection of HSVtk gene driven by AFP promoter inhibits hepatic tumor growth of hepatocellular carcinoma in SCID mice. Gene Ther 8, 80–83 (2001) doi:10.1038/sj.gt.3301355

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Keywords

  • hepatoma
  • alpha-fetoprotein promoter
  • herpes thymidine kinase
  • HVJ-liposome

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