KR100492016B1 - Method for anticancer treatment using adenovirus vector Ad-OA producing antisense telomerase and cisplatin - Google Patents

Abstract

Telomerase functions to add repeat sites to the ends of chromosomes and is known to be involved in cell carcinogenesis through cell immortalization. Therefore, research is being actively conducted to increase the anticancer sensitivity of cancer cells by inhibiting the activity of telomerase. The present invention relates to a method for enhancing the anticancer drug sensitivity of cisplatin, a representative anticancer agent, by using a recombinant adenovirus Ad-OA capable of inhibiting telomerase activity in ovarian cancer cell lines and melanoma cell lines resistant to anticancer drugs. Thus, the potential of an adjuvant drug in anti-cancer drug-resistant tumor cell lines of the recombinant adenovirus Ad-OA.

Description

Anticancer treatment using adenovirus vector Ad-OA producing antisense telomerase and cisplatin}

An object of the present invention is to induce the death of tumor cell lines with anticancer drug resistance by administering a combination of antisense telomerase adenovirus and anticancer agent that can inhibit telomerase activity.

Telomerase does not show activity in most normal cells, but its activity is observed in about 70-95% of tumor cells. That is, 79-100% in breast cancer, prostate cancer, colon cancer, neuroblastoma, hematologic cancer, leiomyosarcoma, rhabdomyosarcoma, 100% of ovarian cancer, 80.1% of lung cancer, 84.8% of stomach cancer, 71.4% of colon cancer, head and neck cancer Almost all carcinomas, including 88.8% of them, showed 80-100% activity. However, the activity of telomerase activity is closely related to cancer because it is hardly found in normal tissues around cancer. Cancer tissues require the activity of telomerase for cell immortalization and are essential for its growth, so inhibition of telomerase activity may be a good target for target-oriented tumor gene therapy. In other words, since telomerase is not observed in normal cells and hardly affects the growth of normal cells, inhibitors that inhibit telomerase activity can overcome the disadvantages of currently used anticancer drugs that act on normal cells. There is this. The recent discovery of several genes that make up human telomerase has been a major turning point in the study of telomerase activity inhibition. That is, several attempts have been made to inhibit human telomerase RNA (hTR) to obtain anticancer effects. According to Bisoffi (Bisoffi et al., 1998), when malignant glioma (MG) cells were infected with an antisense vector against human telomoerase RNA component (hTR), many of the MG cells caused cell death or differentiation. Responded more sensitively to anticancer drugs such as cisplatin and cisplatin. In addition, treatment of retrovirus reverse transcriptase inhibitors to cancer cells resulted in inhibition of telomerase activity and apoptosis, and the same effect in the treatment of various peptide nucleic acids and antisense oligomers against hTR (Bhaswati et al., 1998). , AIGlukhov et al., 1998). Kondo et al. Recently analyzed telomerase mRNA using the MFOLD program to confirm that 94-76 residues were the most open sites. Antisense synthetic oligomers for this site were administered to malignant glioma cells, prostate cancer cells, etc. to obtain a definite cell killing effect, the majority of which was due to the caspase dependent apoptosis (Kondo et al., 1998).

The theory of anticancer therapy using complementary antisense is to prevent the target gene mRNA to be removed using complementary antisense. Methods using such antisense molecules include methods using synthetic antisense oligomers and methods using expressed antisense sequences. However, synthetic antisense oligomers have many problems such as 1) manufacturing difficulties, 2) degradation by nucleases, and 3) the absence of efficient intracellular delivery methods (Scanlon et al., 1995). Therefore, much attention is paid to methods using expression antisense sequences (Lee et al., 1996; Steiner et al., 1998). The principle of this method is to suppress the expression of a specific gene by cloning a cDNA part of the target mRNA in the eukaryotic gene expression vector in the antisense direction to express complementary RNA sequences.

Cisplatin is one of the major anticancer drugs with excellent effects, but its therapeutic effect is still not satisfactory, and the appearance of tumors resistant to cisplatin is an important cause leading to treatment failure. Cisplatin is known to act on DNA and cause apoptosis of cancer cells (Brown R et al., 1997). Despite the breakthrough in the development of anticancer drugs, successful treatments are far from being successful. In addition, excessive use of anticancer drugs reduces blood count, hair loss symptoms, skin rash, effects on the ovaries and testicles, depression, fever, severe constipation or diarrhea, severe pain such as sudden weight loss or weight gain, bleeding and headache, and persistent vomiting. As it causes serious side effects, it is necessary to reduce the amount of anticancer drugs administered to patients.

Telomerase serves to protect the DNA of cells from genetic instability due to chromosomal terminal disruption and to prevent cell death due to this instability. Although telomerase can not be generalized to prevent all DNA damage, inhibition of telomerase can induce aging, differentiation and apoptosis of cancer cells. Since cisplatin induces apoptosis of cancer cells, the inhibition of telomerase plays a role in protecting DNA, and thus, if telomerase can inhibit the telomerase activity of tumor cells, the sensitivity of tumor cells to anticancer agents acting on DNA such as cisplatin You can increase it. In the previous invention, we produced an adenovirus that expresses a region corresponding to 96-74 sites of the telomerase template site in the antisense direction and confirmed its killing effect in lung cancer cell lines and prostate cancer cell lines. By apoptosis. The present invention further relates to the inhibition of telomerase activity and the anticancer effect by cisplatin in melanoma cell lines and ovarian cancer cell lines having anticancer drug resistance.

An object of the present invention is to obtain a tumor-specific anticancer effect using adenovirus Ad-OA that inhibits telomerase activity and cisplatin, an anticancer agent. Telomerase is largely composed of an RNA template region (hTR) and an hTERT region with the activity of reverse transcriptase. To date, research has been reported to have the greatest effect on the inhibition of telomerase RNA template region (hTR). Also, according to Kondo et al., The analysis of telomerase mRNA using the MFOLD program showed that the inhibition of the most open sites (94-76 residues) with synthetic oligomers showed the effect of killing cancer cells (Kondo et al. , 1998). Therefore, in the present invention, by inserting the antisense DNA for the telomerase open hTR site in the adenovirus vector, the anti-cancer effect using the anti-cancer agent cisplatin as a viral vector Ad-OA that inhibits the telomerase activity by the transcribed mRNA Provide a way to get.

The adenovirus clone Ad-OA used in the present invention was deposited on August 29, 2001 to the Korea Microbial Conservation Center (Accession No .: KCCM 10312) and filed with a Korean patent on September 14, 2001 for chemotherapy using the same. (Application number: 10-2001-0057382). Thus, for the preparation of the adenovirus clone Ad-OA, see the above patent application specification.

It is also an object of the present invention to provide a use of the adenovirus clone for the treatment of cancer. The adenovirus clone of the present invention can be particularly useful for lung cancer, prostate cancer and the like. Specifically, the present invention may provide an anticancer gene therapy for treating cancer by inhibiting cancer progression or killing cancer cells by infecting the adenovirus clone to a site where cancer occurs in the human body.

The present invention provides an anticancer treatment method using adenovirus vector Ad-OA expressing antisense telomerase and cisplatin, an anticancer agent.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1 Recombinant Adenovirus Vectors

The recombinant adenovirus vector Ad-OA used in the present invention is an E1a / E1b deficient adenovirus vector, which is inserted into the telomerase template region 94 to 76 in the E1 region of the adenovirus and is the latest promoter of human cytomegalovirus. adenovirus in 293 cell lines using an adenovirus expression vector pΔACMVp (A) comprising an expression early promoter and an expression cassette consisting of late polyadenylation signals of simian virus (SV40). The virus vector was prepared by lipofectin mediated cotransfection with pJM17 (Microbix Biosystems Inc, Toronto, Canada). The virus was propagated in 293 cells. A negative control vector (Ad5.CMV-Null; Ad-CMV) was purchased from Quantum-Appligene (Quantum-Appligene, USA). Ad-CMV is an empty vector and does not have any coding sequence between the CMV promoter and the poly adenylation site (PA).

Example 2 Cell Line Culture

Ovarian cancer cell line A2780S and anticancer drug resistant A2780 / cp70 cell line, melanoma cell line A375P, and DM4 cell line in RPMI 1640 (GIBCO BRL, Gaithersburg, MD) with 2 mM L-glutamine (GIBCOBRL, Gaithersburg, MD) and 10% fetal bovine The medium containing serum (GIBCO BRL, Gaithersburg, MD) and 1% penicillin streptomycin (GIBCO BRL, Gaithersburg, MD) was incubated at 37 ° C and 5% CO ₂ . Melanoma cell lines were used by the Korea Cell Line Bank for ovarian cancer cell lines from Dr. Dr. Robert Brown of the University of Glasgow, UK.

Example 3 IC ₅₀ for Cisplatin for Each Cell Line

The same number of cells were incubated in 96 well plates for 24 hours, treated with various concentrations of Cis-dichlorodiammineplatinum (cisplatin; Sigma, US), and then cultured again for 72 hours, followed by Cytotoxic assay (MTT assay). After incubation, 50 µl of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide (MTT; Sigma, US) solution (2mg / ml) was added thereto, and the conditions were 37 ° C and 5% CO ₂ . Incubated for 4 hours under. 100 μl of DMSO was added thereto, and the resultant was allowed to stand for 15 minutes. Then, the absorbance was measured by ELISA reader (540 nm) to determine the IC ₅₀ value. As a result, ovarian cancer cell line A2780S and anticancer drug-resistant cell line A2780 / cp70 showed IC ₅₀ in 8 μM and 32 μM, respectively, and melanoma cell lines in A375P and DM4 at about 80 μM cisplatin.

Example 4 Effect of Ad-OA on Cancer Cell Killing by Cisplatin

First, the most effective dosing method was determined using A2780 / cp. In other words, the first A2780 / cp infection with 100 moi Ad-OA for 2 days, then the cisplatin administration method, the second treatment with cisplatin 2 days after Ad-OA infection method, third Ad-OA and cisplatin at the same time The method of administration was used. As a result, the first method of cisplatin treatment after Ad-OA infection observed about 80% cell growth inhibition effect compared to the mock test (FIG. 1). Therefore, other tumor cell lines were also treated in this way. That is, after 48 hours after Ad-OA treatment with 100 moi of each cell line, cisplatin at the concentration corresponding to IC ₅₀ of each cell line was treated 6 days after MTT assay. It was confirmed (FIGS. 2, 3).

When the adenovirus Ad-OA and cisplatin of the present invention are used together, it can be confirmed that the anti-cancer drug-resistant cancer cells have an excellent effect on killing or inhibiting growth. In addition, the method is expected to be anti-cancer gene therapy that minimizes the effect on the patient by minimizing the side effects caused by excessive use of anticancer drugs.

1 is a result of treatment with 32 μM cisplatin after Ad-OA 100 moi infection with an anticancer drug-resistant ovarian cancer tumor cell line A2780 / cp70.

Lane 1: Mock Experiment

Lane 2: Ad-CMV Infection

Lane 3: Ad-OA Infection

Lane 4: cisplatin treatment after Ad-OA infection

Lane 5: Ad-OA infection after cisplatin treatment

Lane 6: Simultaneous Ad-OA and Cisplatin

Figure 2 shows the results of 80 μM cisplatin treatment after infection with melanoma cell lines A375-P, DM4 100 Ad-OA moi.

Lane 1: Ad-CMV Infection

Lane 2: Ad-OA Infection

Lane 3: cisplatin treatment

Lane 4: Cisplatin Treatment After Ad-OA Infection

Claims (1)

An anticancer agent comprising adenovirus clone Ad-OA (Accession No .: KCCM 10312) which inhibits telomerase activity and an anticancer agent cisplatin.