CN106822085B

CN106822085B - Application of oncolytic adenovirus expressing TRAIL and quercetin in inhibition of liver cancer cell proliferation

Info

Publication number: CN106822085B
Application number: CN201611171522.3A
Authority: CN
Inventors: 邹海; 黄东胜; 童向民; 王世兵; 牟晓洲
Original assignee: Zhejiang Provincial Peoples Hospital
Current assignee: Zhejiang Provincial Peoples Hospital
Priority date: 2016-12-17
Filing date: 2016-12-17
Publication date: 2020-03-17
Anticipated expiration: 2036-12-17
Also published as: CN106822085A

Abstract

The invention relates to the field of biological pharmacy, in particular to application of oncolytic adenovirus for expressing TRAIL and quercetin in inhibiting proliferation of liver cancer cells. Specifically, the invention relates to a combination of oncolytic adenovirus containing TRAIL, quercetin and other medicines, which has synergistic treatment or inhibition effect on liver cancer or proliferation of liver cancer cells, and further provides a new composition or a medicine box for treating liver cancer or inhibiting proliferation of liver cancer cells.

Description

Application of oncolytic adenovirus expressing TRAIL and quercetin in inhibition of liver cancer cell proliferation

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to application of oncolytic adenovirus expressing TRAIL in combination with quercetin in inhibition of liver cancer cell proliferation.

Background

Hepatocellular carcinoma (HCC) is one of the most common malignancies, classified as the fifth most common cancer worldwide with annual mortality rates exceeding 50 ten thousand.

PCT/US2014/033675 discloses methods and compositions for treating various cancers, including primary and secondary liver cancers, while relating to the use of quinacrine in the manufacture of a medicament for refractory liver cancers.

PCT/US2008/081645 provides a method for treating liver cancer. These methods comprise administering a compound comprising a modified oligonucleotide, wherein the modified oligonucleotide targets a miRNA. The invention also provides a composition for treating liver cancer. Such compositions include compounds comprising modified oligonucleotides, wherein the modified oligonucleotides are targeted to mirnas. The invention finds that certain mirnas are overexpressed in liver cancers (e.g., hepatocellular carcinoma) and are therefore selected to be targeted by modified oligonucleotides. In addition, the invention also finds that certain mirnas are overexpressed in hepatocellular carcinoma cells exposed to dioxin, and are therefore selected to be targeted by modified oligonucleotides.

201080047449.3 discloses a method of interventional treatment or eradication of cancer by administering an effective amount of an endogenous ligand for the aryl hydrocarbon (Ah) receptor (AhR) designated ITE or one of its analogs (active ingredient) to a subject suffering from cancer. The effective dose and frequency of administration are determined by measuring the blood level of the active ingredient in the subject after administration. The active ingredient formulated with the carrier system is applied topically, enterally or parenterally to the subject. The formulated drug may also be administered with one or more other cancer therapeutic agents. After the subject is free of cancer, a maintenance dose is provided to ensure eradication of the cancer. Wherein the subject having cancer such as liver cancer is preferably treated.

Adenovirus (Ad) was first discovered by Wallace Rowe and co-workers in 1953. Due to the cytopathic effect of Ad in tissue culture, the virus was used in clinical trial studies for the treatment of cervical cancer in 1956. The use of oncolytic viruses has yielded excellent therapeutic efficacy over the last two decades. Most clinical and preclinical studies have focused on oncolytic virus modification to improve tumor transduction targeting, tumor specific replication, intratumoral dissemination, and modulation of antiviral and antitumor immune responses as well as the transfer of foreign genes. Modification of oncolytic viruses and hybrid engineering of oncolytic viruses are particularly promising new approaches to cancer therapy. Viral therapy using oncolytic Ad has widespread clinical applications due to its high titer, ability to insert therapeutic genes of larger size and high transduction efficiency in dividing and non-dividing cells. Importantly, when oncolytic ads replicate, they do not integrate their genome into the host; thus, they do not induce carcinogenesis-related mutagenesis. These unique functions allow oncolytic Ad to be an efficient gene vector with optimal safety in gene delivery (compared to related oncolytic viruses such as oncolytic retroviruses, lentiviruses and adeno-associated viruses).

Currently, more than 12 different oncolytic viruses are undergoing phase I clinical trials for different types of cancer. Oncolytic Ad is the first and most intensively studied oncolytic virus to date, H101 (a mutant lacking the E1B55K gene) has now been approved by the Chinese Food and Drug Administration (CFDA) for the treatment of head and neck cancer (Oncorine, Shanghai Shuangwei Biotech). In previous studies, we developed cancer-targeting genes (CTGVT) using viral therapy strategies and generated a new E1B55K gene-deleted oncolytic adenovirus ZD55-TRAIL, which carries a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is a member of the tumor necrosis factor superfamily and is considered as a new candidate for anti-cancer therapy. TRAIL can selectively induce apoptosis of various tumor cells, and has negligible toxicity to surrounding normal cells. The selectivity of TRAIL in inhibiting tumor growth has been shown in vitro and in vivo. In contrast, TRAIL can activate the transcription factor NF-. kappa.B, leading to the transcription of genes that antagonize the death signaling pathway. Unfortunately, due to this opposite or regulatory effect exerted by TRAIL, many cancer cells also show resistance to TRAIL despite its dramatic anti-tumor activity. The actual mechanism by which TRAIL participates in this negative feedback regulation of apoptotic signaling through NF- κ B has not yet been fully elucidated. Fortunately, recent studies have shown that combination therapy with TRAIL and other drugs or chemicals can synergistically increase cell death or apoptotic mechanisms in a variety of tumor cells by inhibiting survival signaling and expression of proliferative genes while activating apoptosis-related gene expression.

Quercetin (3,3', 4', 5, 7-pentahydroxyflavone) is the major dietary flavonol found in a variety of fruits and vegetables, including onion, apple peel, lettuce, cauliflower, capsicum, celery, and unsweetened cocoa. Quercetin has antioxidant, anti-inflammatory, anti-angiogenic, anti-proliferative and pro-apoptotic properties and, therefore, can combat the development and progression of cancer. According to these effects, quercetin causes a reduction in cell viability through its ability to selectively modify the signaling pathways associated with carcinogenesis to cause cancer cell death, and induces apoptosis in many cancer cells (including breast, colon, lung, ovarian and prostate cancers).

Disclosure of Invention

The inventor of the present invention proves for the first time that ZD55-TRAIL and quercetin can kill HCC cells in a synergistic effect in vitro and in vivo. Furthermore, our findings indicate that quercetin significantly inhibits the NF κ B pathway activated by ZD55-TRAIL, such that the apoptotic signal dominates. Our current research shows that the combination therapy of ZD55-TRAIL and quercetin is a new practical therapeutic strategy.

In order to better cure liver cancer or provide another feasible option for treating liver cancer and improve the prognosis or the life quality of patients, the inventor finds that the combination of oncolytic adenovirus expressing TRAIL and quercetin has synergistic treatment or inhibition effect on liver cancer or proliferation of liver cancer cells, thereby providing a new treatment method or means for treating liver cancer or inhibiting proliferation of liver cancer cells.

The invention adopts the oncolytic adenovirus expressing TRAIL and the quercetin to jointly inhibit the proliferation effect of the liver cancer cells to carry out in vitro and in vitro experiments, and the result shows that the oncolytic adenovirus expressing TRAIL and the quercetin jointly applied have the effect of synergistically inhibiting the proliferation of the liver cancer cells.

The oncolytic adenovirus for expressing TRAIL can be replicative oncolytic adenovirus ZD55-TRAIL (Han, X., et al., Synergistic combination of histone deacetylase subunit and suberoylanilide hydroxamic acid and oncocytic adenovirus ZD55-TRAILas a viral antigen cancer. mol Med Rep,2015.12 (1)) carrying TRAIL gene, has tumor replication specificity and has no toxicity to normal cells. The genome structure of oncolytic adenovirus expressing TRAIL is shown in figure 1A.

Accordingly, in one aspect, the present invention provides the use of an oncolytic adenovirus expressing TRAIL for the preparation of a pharmaceutical composition or kit for the treatment of liver cancer or inhibition of liver cancer cell proliferation in a subject, wherein the pharmaceutical composition or kit comprises an oncolytic adenovirus expressing TRAIL and quercetin, optionally further comprising other pharmaceutically active ingredients for the treatment of liver cancer or inhibition of liver cancer cell proliferation, such as gemcitabine, plerixafor (AMD3100), paclitaxel, or the like. Optionally, the oncolytic adenovirus expressing TRAIL and quercetin in the pharmaceutical composition or kit are stored in separate containers or the oncolytic adenovirus expressing TRAIL and quercetin are stored in the same container.

The present invention provides a method of treating liver cancer or inhibiting proliferation of liver cancer cells in a subject comprising administering to the subject, simultaneously or sequentially, an oncolytic adenovirus that expresses TRAIL and quercetin. Optionally, in the method of the present invention, other pharmaceutically active ingredients for treating liver cancer or inhibiting proliferation of liver cancer cells, such as gemcitabine, plerixafor (AMD3100), paclitaxel, or the like, may be administered simultaneously or sequentially; quinidine is administered either simultaneously or sequentially.

For the subject of the present invention, it is preferably a mammal, more preferably a human.

For the TRAIL expressing oncolytic adenoviruses and quercetin of the present invention, one skilled in the art can make any modification thereto, provided that the modification does not negatively affect its activity. For example, the compound may be modified or loaded onto other carriers to increase its half-life in vivo; or may be linked to known penetration peptides to facilitate transdermal absorption of the compounds of the invention or crossing the blood brain barrier, etc. In general, various modifications can be made to the compounds of the invention by those skilled in the art to increase the efficiency of delivery or for other purposes and to maintain their activity. For modification of the virus, for example, one skilled in the art may make genetic modifications to express additional active ingredients against cancer. Such modifications are also within the scope of the present invention.

The oncolytic adenovirus expressing TRAIL and quercetin of the present invention as active ingredients may be used together with a pharmaceutically acceptable carrier. The methods, uses and products of the invention may comprise, in addition to the active ingredient, suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active ingredient into preparations, for example suitable for injection or infusion.

Formulations suitable for injection or infusion may include aqueous and non-aqueous sterile injection solutions, which may optionally contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

The active ingredients of the invention may optionally be combined with solid excipients and the resulting mixture optionally ground and, if desired, after addition of suitable auxiliaries, processed to give the desired dosage form. Suitable excipients are in particular fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose or starch preparations, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate, may be added.

The amount of oncolytic adenovirus expressing TRAIL and quercetin administered in the present invention may be any amount that synergistically treats liver cancer or inhibits proliferation of liver cancer cells in a subject, and may be a dose equivalent to about 0.2-25mg of quercetin, preferably 0.02-30mg of quercetin and 1-100MOI of oncolytic adenovirus expressing TRAIL. More preferably, the dosage unit comprises about 1-5mg of quercetin and 20-60MOI of an oncolytic adenovirus that expresses TRAIL. Most preferably, the dosage unit comprises about 2-3mg of quercetin and 40-50MOI of an oncolytic adenovirus expressing TRAIL. Determination of an effective amount is within the ability of those skilled in the art, particularly in light of the disclosure provided herein.

According to the present invention, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the present invention may be administered to a subject in any effective dose. Preferably, the pharmaceutical product (drug, medicament or kit) or pharmaceutical composition of the invention may be administered in multiple doses, for example from about 2 to about 15 doses, more preferably from about 4-10 doses, most preferably about 6 doses. In a particularly preferred embodiment, the pharmaceutical product (drug, medicament) or pharmaceutical composition of the invention is administered to the subject during the course of administration, e.g. injection, infusion or oral administration, at a frequency of about once every three weeks. In a particularly preferred embodiment, the administration is by intravenous injection of quercetin, by intraperitoneal injection of an oncolytic adenovirus expressing TRAIL.

It will be appreciated that the pharmaceutical product (medicament, medicament or kit) or pharmaceutical composition of the invention may be formulated in any suitable manner for administration by any suitable route.

Dosage units of the pharmaceutical products (drugs, medicaments) or pharmaceutical compositions of the invention are based on conventional administration to a subject. For example, a dosage unit may be administered more than once daily, once weekly, once monthly, etc. Dosage units may be administered on a twice/week basis, i.e., twice weekly, e.g., once every three days.

As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. The term "comprising" in any of the expressions herein, particularly in describing the method, use or product of the invention, is to be understood as including those products, methods and uses which consist essentially of and consist of the recited components or elements or steps. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

Instructions relating to the pharmaceutical product (e.g., pharmaceutical composition or kit) may be included in the pharmaceutical product of the invention, and may include the following: indications (e.g., liver cancer), dosages administered (e.g., as exemplified above), and possible side effects, among others.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

For a more clear illustration of the invention, reference is now made in detail to the following examples, which are intended to be purely exemplary of the invention and are not to be interpreted as limiting the application.

Drawings

FIG. 1: characterization of ZD 55-TRAIL. (A) Schematic structure of ZD 55-TRAIL. In ZD55-TRAIL, the E1B 55-kDa gene was replaced by an expression cassette for SV40polyA and TRAIL. (B) ZD55-TRAIL was identified by western blot analysis. HuH-7 cells were treated with quercetin (10. mu.M), ZD55-TRAIL (2MOI), or quercetin (10. mu.M) and ZD55-TRAIL (2MOI), respectively. After 48 hours, cell lysates were prepared for analysis of TRAIL and E1A protein expression. Mock infected cells served as controls. GAPDH was used as protein loading control.

FIG. 2: quercetin enhances ZD 55-TRAIL-mediated inhibition of HCC cell growth. (A) HCC cell lines HepG2, HuH-7 and SMMC-7721 were treated with ZD55-TRAIL (1,2,5,10MOI), quercetin (5,10,25, 50. mu.M) or a combination thereof, respectively, for 48 hours. The figure shows exemplary results of three independent experiments. Cell viability was assessed by MTT assay. (B) The synergistic effect of ZD55-TRAIL in combination with quercetin on HCC cells. Quantification was done by Combination Index (CIN) analysis and expressed as 1g (CIN) to affected ratio. Where calculations are possible, a 95% confidence interval is displayed.

FIG. 3: Quercetin enhances apoptosis of HCC cells induced by ZD55-TRAIL α (A) apoptosis was detected using Hoechst 33342 staining HuH-7 was plated in 96-well plates and infected with ZD55-TRAIL (2MOI), Quercetin (10 μ M), or ZD55-TRAIL (2MOI) plus Quercetin (10 μ M). to determine the extent of apoptosis, HuH-7 cells were treated with Hoechst 33342 for 30 minutes 72 hours after treatment and then observed under an inverted fluorescence microscope.Red arrows indicate positive apoptotic cells.original magnification was 400. (B) HuH-7 cells were treated with ZD55-TRAIL (2MOI), Quercetin (10 μ M), or ZD55-TRAIL (2MOI) plus Quercetin (10 μ M). after 48 hours untreated cells were treated with control, apoptosis was determined by flow cytometry (C) using ZD55-TRAIL (2MOI), or ZD55-TRAIL (10 MOI) plus Quercetin (10 μ M). Suitabine was prepared for full-amplification assay using a full blot of the whole cell assay.

FIG. 4 inhibition of NF- κ B activation in HCC cells induced by ZD55-TRAIL by Quercetin HuH-7 cells were treated with ZD55-TRAIL (2MOI), Quercetin (10 μ M) or ZD55-TRAIL (2MOI) plus Quercetin (10 μ M). after 48 hours, cell lysates were collected and subjected to Western blot assays to check for changes in Iκ B α, p65 and p 50. GAPDH expressed was used as a loading control.

FIG. 5: ZD55-TRAIL and Quercetin. (A) Tumor volumes were measured at different times after treatment. Data are presented as mean ± SD (n ═ 6). P < 0.01. (B) The figure shows the inhibition of tumor growth for each group at the last time point (day 55) of mice sacrifice.

Detailed Description

The invention is further explained by the accompanying drawings and examples.

The materials and methods used in the examples of the invention of the present application are as follows.

Cell lines and viruses

Human hepatoma cell lines SMMC-7721, HepG2 and HuH-7 were purchased from cell culture Collection cell Bank (CBTCCAS, Shanghai, China) of China academy of sciences and cultured in Dulbecco's modified Eagle Medium (DMEM, GIBCO, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO, Carlsbad, CA). Cells were incubated at 37 ℃ in 5% CO₂Incubation in a humid incubator. Construction of recombinant oncolytic adenovirus ZD55-TRAIL and its useThe production is as described above. Amplification of recombinant adenovirus was performed by infection of HEK293 cells.

In vitro synergistic cytotoxicity assays and quantitation

3- (4, 5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (StLouis, Mo.). Cell viability was obtained by MTT assay as previously described. Briefly, 1 × 10⁴Individual SMMC-7721, HepG2 or HuH-7 cells were seeded in 96-well plates and treated with ZD55-TRAIL and quercetin at the indicated concentrations. 48 hours after treatment, 10. mu.L of MTT solution (5g/L) was added to each well and incubated at 37 ℃ for 4 hours. The absorbance at 570nm was measured using a DNA microplate reader (Tecan, Maennedorf, Switzerland).

Hoechst 33342 staining

Hoechst 33342 staining was used to detect apoptotic changes. HuH-7 cells were treated with ZD55-TRAIL, quercetin or a combination of ZD55-TRAIL and quercetin, respectively. After 72 hours of treatment, 5. mu.L of Hoechst 33342(1mg/ml, Sigma-Aldrich, St Louis, Mo.) was added to the cells for 30 minutes and the results were observed under an inverted fluorescent microscope. Untreated cells were used as controls.

Flow cytometry analysis

HuH-7 cells were treated with ZD55-TRAIL (2MOI), quercetin (10. mu.M), or ZD55-TRAIL (2MOI) and quercetin (10. mu.M). After 48 hours, apoptotic cells were detected alone using Annexin V-FITC and PI double staining or PI staining according to the instructions. Apoptosis and cell cycle progression were examined using flow cytometry (FACStar cytofluorometer, BD Biosciences).

Western blot analysis

HuH-7 cells were collected and washed twice with PBS, then lysed in RIPA buffer the proteins were isolated by loading the Cell extracts on a 12% SDS-polyacrylamide gel and subsequently transferred to PVDF (polyvinylidene fluoride) membranes the membranes were then incubated with primary antibodies and dilutions directed against adenovirus-5E 1A (1:1000), TRAIL (1:1000), Capase-9(1:1000), Caspase-3(1:1000), PARP (1:1000), GAPDH (1:1000), p65(1:500), p60(1:500) and I κ B α (1: 500). the secondary antibodies used and their dilutions were anti-rabbit (1:5000) and anti-mouse (1:5000), anti-adenovirus-5E 1A, Caspase-9, Caspase-3, PARP and GAPDH were purchased from Sanuz Biotechnology (Sanpasy), Sanpase-5, Cell II, Cell 3875, Cell II, Cell III, Cell 3875, Cell III, and Cell III antibodies from Siguz Biotechnology (1: 5000).

Animal experiments

The permission for animal experiments was granted by the animal protection and use committee of china. Animals and experiments were performed according to the criteria of the above mentioned institutions. 5-week-old male BALB/C nude mice were purchased from Shanghai laboratory animal center, national academy of sciences, Shanghai, China. HuH-7 cells were injected subcutaneously into the right flank of male nude mice. Mice were examined three times per week to observe tumor development. Once the subcutaneous tumor develops and reaches about 100-³Nude mice were randomly divided into four groups (6 mice per group). Subsequently, mice were injected with ZD55-TRAIL, quercetin, and the combination of the virus and the drug or PBS, respectively. ZD55-TRAIL (1X 10) administered by intratumoral injection⁹Plaque forming unit-PFU, per mouse) while administering 100 μ Ι pbs vehicle to the parallel control by intragastric administration of quercetin to mice at a dose of 150mg/kg body weight or by injection or by intragastric administration. Tumor length and width (mm) were measured every 3 days using a vernier caliper, and tumor volume was calculated using the following formula: tumor volume (mm)³)＝(A×B²) And/2, wherein A and B represent length and width, respectively. Tumor growth curves were then obtained from the volume measurements.

Statistical analysis

Experimental statistical significance was expressed as mean. + -. Standard Deviation (SD) and analyzed by GraphPad 6.0 Software (GraphPad Software, San Diego, Calif.). Student's t-test was performed to determine statistical significance. P values <0.05 were considered statistically significant.

Example 1: quercetin enhances ZD 55-TRAIL-mediated inhibition of HCC cell growth.

An oncolytic adenovirus vector ZD55 is constructed by deleting the E1B 55-kDa gene of adenovirus 5; it can selectively replicate in many tumor cells. Based on the cancer-targeting gene-Viro-Therapy, we used ZD55 to package the therapeutic gene TRAIL and to obtain a novel recombinant oncolytic adenovirus ZD55-TRAIL (figure 1A). To verify the characterization of ZD55-TRAIL, we used western blot analysis to detect protein expression of adenovirus E1A and the therapeutic gene TRAIL. The presence of strong expression of E1A and TRAIL in HuH-7 cells infected with ZD55-TRAIL and ZD55-TRAIL plus quercetin, compared to control cells treated without ZD55-TRAIL, indicates that ZD55-TRAIL can selectively replicate FIG. 1B at high levels in HCC cells).

To assess whether quercetin enhances ZD55-TRAIL induced cell death of HCC cells, MTT assays were performed as described in materials and methods. HCC cell lines SMMC-7721, HepG2 and HuH-7 were infected with ZD55-TRAIL alone, quercetin alone or a combination of both. The results show that the combined treatment with ZD55-TRAIL and quercetin resulted in enhanced tumor killing effect on HCC compared to control cell lines (figure 2).

Example 2: quercetin enhances ZD55-TRAIL to induce apoptosis of HCC cells.

Hoechst 33342 staining was then performed to determine the morphological changes of HuH-7 cells treated with ZD55-TRAIL plus quercetin, ZD55-TRAIL or quercetin alone. The results are shown in FIG. 3A. This demonstrates that co-treatment with quercetin resulted in significant apoptosis characterized by chromatin condensation, nuclear fragmentation and apoptotic bodies observed compared to ZD55-TRAIL alone treatment. To quantify the effect of quercetin and ZD 55-TRAIL-induced apoptosis, annexin-V-Fluorescein Isothiocyanate (FITC)/PI double staining was used to analyze apoptosis (fig. 3B). The results showed that the HuH-7 apoptosis rate with ZD55-TRAIL and quercetin was 58.9%, almost twice that of ZD55-TRAIL alone (27.4%).

Western blot analysis demonstrated that ZD55-TRAIL has the ability to activate caspase-dependent pathways, including caspase-9, caspase-3 activation and PARP cleavage. This unique effect may be further enhanced by co-treatment with quercetin and ZD55-TRAIL (figure 3C).

Example 3: quercetin inhibits activation of NF-kB in HCC cells induced by ZD55-TRAIL

To demonstrate the ability of quercetin to inhibit NF-. kappa.B-dependent transcription and subsequently sensitize HCC cells to ZD55-TRAIL, we used Western blotting to determine the changes in expression of I.kappa.B α, p65 and p50 caused by the treatment as shown in FIG. 4, the reduced expression of IkBa, p65 and p50 in the combined treatment of quercetin and ZD55-TRAIL was more pronounced than that induced by ZD55-TRAIL alone, which indicates that quercetin can indeed reduce ZD 55-TRAIL-mediated activation of NF-. kappa.B and reduce its transcriptional activity by promoting ZD 55-induced apoptosis (FIG. 4).

Example 4: quercetin enhances ZD 55-TRAIL-mediated inhibition of tumor growth in HCC in vivo

To test the therapeutic effect of combined quercetin and ZD55-TRAIL treatment in vivo, animal experiments were conducted with single and combination therapeutic treatments using an HCC tumor xenograft model established with HuH-7 cells. Tumor growth curves were plotted to compare the difference in antitumor efficacy over the 49 day observation period. As shown in the figure. As shown in FIG. 5A, mean tumor volumes were significantly reduced in intratumorally injected animals receiving quercetin alone, ZD55-TRAIL and combination treatment compared to those receiving PBS injection. As expected, the combination treatment was more effective than quercetin (P ═ 0.001) and ZD55-TRAIL alone (P ═ 0.002). Furthermore, the combination treatment of quercetin and ZD55-TRAIL resulted in improved animal survival compared to vehicle, quercetin or ZD55-TRAIL alone treatment (figure 5B).

Example 5: ZD55-TRAIL, quercetin and quinidine can obviously inhibit the growth of liver cancer cells in vivo

4-5 weeks old female BALB/c nude mice were purchased from Shanghai laboratory animal center (Shanghai, China), academy of sciences, China. All experimental procedures were in accordance with welfare and ethical requirements of experimental animals. HuH-7 cell (2X 10)⁶In PBS) were injected subcutaneously on the right side of nude mice. Once the subcutaneous tumor size reaches 100-³Nude mice were randomly divided into 3 groups (8 mice per group). Then the combination of quercetin and ZD55-TRAIL, and the combination of quercetin, ZD55-TRAIL and quinidine (Quin) are administered. ZD55-TRAIL (1X 10)⁹PFU/mouse) was injected intratumorally, while quercetin and quinidine were injected intraperitoneally at 5mg/Kg body weight, with 100 μ l PBS as control, three consecutive injections, every other day. Tumor size was measured every 5 days from the start of injection and survival of nude mice was counted over a period of 75 days。

The results confirmed that the combination of quercetin and ZD55-TRAIL and the combination of quercetin, ZD55-TRAIL and quinidine were able to substantially eliminate tumor-bearing mass of nude mice at day 50, but the combination of quercetin and ZD55-TRAIL resulted in a survival rate of 62.5% (3 nude mice died) and the survival rate of the combination of quercetin, ZD55-TRAIL and quinidine of 87.5% (1 nude mouse died) at day 75 at the end of the experiment.

Although the present invention has been described in the above-mentioned embodiments, it is to be understood that the present invention may be further modified and changed without departing from the spirit of the present invention, and that such modifications and changes are within the scope of the present invention.

Claims

1. Use of an oncolytic adenovirus that expresses TRAIL, quercetin and quinidine, in the preparation of a pharmaceutical composition or kit for treating liver cancer or inhibiting proliferation of liver cancer cells in a subject, wherein the pharmaceutical composition or kit is a pharmaceutical composition or kit for injection.

2. The use according to claim 1, wherein the pharmaceutical composition or kit comprises an oncolytic adenovirus that expresses TRAIL, quercetin and quinidine, each in separate containers or in the same container.