CN106860460A - Applications of the glutaminase inhibitor C B 839 in the medicine for preparing treatment estrogen sensitive type carcinoma of endometrium - Google Patents

Abstract

The invention relates to the application of CB-839, a glutaminase inhibitor, in the preparation of drugs for treating estrogen-sensitive endometrial cancer. The present invention has been confirmed by in vitro experiments and in vivo animal experiments that CB-839 can effectively inhibit the enhancement of glutamine metabolism level of endometrial cancer caused by estrogen stimulation, thereby inhibiting glutamine metabolism in endometrial cancer cells, which is beneficial to limit Oral administration of CB‑839 can significantly reverse the increased glutamine metabolism in endometrial cancer caused by abnormal estrogen levels. Therefore, CB‑839 can be used to prepare drugs for treating estrogen-sensitive endometrial cancer.

Description

Glutaminase Inhibitor CB-839 in the Treatment of Estrogen Sensitive Endometrium cancer drug applications

technical field

The invention belongs to the technical field of medicine and relates to the application of CB-839, a glutaminase inhibitor, in particular to the application of CB-839 in the preparation of drugs for treating estrogen-sensitive endometrial cancer.

Background technique

Endometrial cancer (hereinafter referred to as endometrial cancer) tends to occur in perimenopausal and postmenopausal women, and is one of the most common malignant tumors of the female reproductive system. In 2018, there were 63,400 new cases of endometrial cancer in China, with 21,800 deaths, ranking the second among malignant tumors of the female reproductive system and the highest incidence in Beijing and Shanghai, seriously endangering women's health.

It is known that estrogen is the main pathogenic factor of endometrial cancer. As a soluble mediator, estrogen acts on various stages of endometrial cancer initiation, inflammation and progression, and participates in the pathogenesis of receptor- and non-receptor-dependent endometrial cancer. However, in the clinical diagnosis and treatment process, the estrogen-targeted regimen has not achieved the expected effect, and the 5-year survival rate of advanced endometrial cancer is only 5% to 25%. Studies have found that estrogen has an important effect on the metabolism of proteins and nucleic acids, especially in sexual organs, suggesting that estrogen may promote the process of endometrial cancer through metabolic pathways. However, the mechanism of estrogen metabolism regulation in endometrial cancer is still unclear so far. Therefore, the research on the regulation of estrogen metabolism in endometrial cancer is facing major challenges and opportunities. Intervention strategies are yet to be established.

Glutamine is an important metabolite that has an important effect on cell growth. However, the effect of glutamine on the autophagy and progression of endometrial cancer cells and its related mechanisms are far from clear.

Autophagy is a ubiquitous self-phagy phenomenon in eukaryotic cells. Studies have found that the level of autophagy in cancer cells such as breast cancer, liver cancer, and ovarian cancer is lower than that of normal cells. Autophagy has different mechanisms of action in different stages of tumor development. Most scholars believe that the increase of autophagy activity can inhibit the occurrence and development of tumors. At present, it is believed that the role of autophagy in inhibiting tumors mainly has the following two aspects: on the one hand, the occurrence of autophagy causes cells to enter autophagic death. On the other hand, a variety of oncogenes and tumor suppressor genes play an anti-tumor role by participating in the autophagy pathway, such as Beclin1, PTEN, Bcl-2, etc. Therefore, the abnormal regulation of autophagy is directly related to the occurrence and development of tumors.

CB-839 is a potent, selective and orally active inhibitor of glutaminase. The molecular weight is 571.57, the molecular formula is C ₂₆ H ₂₄ F ₃ N ₇ O ₃ S, the CAS number is 1439399-58-2, and the structural formula is as follows:

At present, there is no report on the therapeutic effect of CB-839 on endometrial cancer.

Contents of the invention

The purpose of the present invention is to provide a use of glutaminase inhibitor CB-839.

The first aspect of the present invention provides the application of CB-839, a glutaminase inhibitor, in the preparation of a drug for treating estrogen-sensitive endometrial cancer.

Another aspect of the present invention provides the application of glutaminase inhibitor CB-839 in the preparation of preparations, and the reagent is used for:

a) inhibit glutamine metabolism in endometrial cancer cells in vitro,

b) promote endometrial cancer cell autophagy in vitro,

c) inhibit endometrial cancer cell proliferation in vitro,

d) inhibiting estrogen-induced proliferation of endometrial cancer cells in vitro, and/or

e) In vitro reversal of estrogen-induced inhibition of endometrial cancer cell autophagy.

As a specific embodiment of the present invention, the endometrial cancer cells are Ishikawa or KLE cells.

The present invention studies the inhibitory effect of glutaminase inhibitor CB-839 on estrogen-induced glutamine metabolism in estrogen-sensitive endometrial cancer in vitro, and further evaluates the therapeutic effect of CB-839 through in vivo experiments. The value of estrogen-stimulated abnormal proliferation of endometrial cancer, the results show that: 1) estrogen promotes the expression of glutaminase in hormone-sensitive endometrial cancer and enhances the metabolism of glutamine; 2) high levels of glutamine Amide metabolism inhibits the autophagy of endometrial cancer cells and promotes the proliferation of endometrial cancer cells; 3) Estrogen can reduce the autophagy level of endometrial cancer and promote the proliferation of endometrial cancer, but the glutaminase inhibitor CB- After 839, the growth of endometrial cancer was significantly restricted and the level of autophagy was increased. It is suggested that the glutaminase inhibitor can be further used in the preparation of drugs for treating estrogen-sensitive endometrial cancer.

Based on this, the present invention provides a treatment method for estrogen-sensitive endometrial cancer using glutaminase inhibitor CB-839, which can inhibit endometrial cancer caused by estrogen by oral administration of glutaminase inhibitor CB-839. The level of cancer autophagy is reduced, thereby improving the progression of endometrial cancer caused by abnormal estrogen.

The invention provides a new method and a new idea for the clinical treatment of endometrial cancer.

Description of drawings

Figures 1-6. Estrogen promotes the proliferation of estrogen-sensitive endometrial cancer cell lines and inhibits autophagy.

Among them, ER-α: estrogen receptor α,

Ishikawa: Estrogen-sensitive endometrial cancer cell line,

KLE: Estrogen-insensitive endometrial carcinoma cell line,

Actin: actin as an internal reference protein,

OD value: absorbance value,

Fulvestrant: Fulvestrant, an estrogen receptor inhibitor,

LC3B-I, LC3B-II: autophagy microtubule-associated protein light chain β3-I, autophagy microtubule-associated protein light chain β3-II,

Beclin-1: a homolog of the yeast autophagy gene Atg6/Vps30,

p62: a nucleoporin negatively associated with the extent of autophagy,

*P<0.05, **P<0.01; ns: no significant difference.

Figures 7-10 show that estrogen promotes glutamine metabolism in estrogen-sensitive endometrial cancer cell lines.

Among them, Glutaminase: glutaminase,

Glutamine; Glutamine,

**P<0.01.

Figures 11-16 show that glutamine metabolism inhibits endometrial cancer autophagy and promotes endometrial cancer proliferation.

Among them, CB-839: is a selective glutaminase inhibitor,

Glutamine: 200mM glutamine treatment group,

Glutamine+CB-839: Glutamine and CB-839 treatment group at the same time,

*P<0.05, **P<0.01, ***P<0.001; ns: no significant difference.

Figure 17-20 shows the inhibitory effect of CB-839 on subcutaneous endometrial cancer xenografts in nude mice.

Among them, Isotype: negative control group,

Control: Tumors in nude mice without drugs,

CB-839: tumors in nude mice orally administered CB-839,

Ki-67 is a nuclear antigen specifically associated with cell proliferation,

Tumor volume calculation formula: volume = (length * width ² )/2,

**P<0.01, ***P<0.001.

Figures 21-25 are CB-839 inhibiting estrogen-promoting glutamine metabolism.

Among them, Estrogen: estrogen treatment group,

Estrogen+CB839: Simultaneous treatment group of estrogen and CB-839,

*P<0.05, **P<0.01; ns: no significant difference.

Figures 26-30 are nude mice orally administered CB-839 to inhibit the growth-promoting effect of estrogen on estrogen-sensitive endometrial cancer.

Among them, Control: Tumor-forming nude mice that were neither administered estrogen nor administered CB-839,

Estrogen: Estrogen-applied tumor-forming nude mice,

Estrogen+CB-839: Tumor-forming nude mice treated with estrogen and CB-839 at the same time,

*P<0.05, **P<0.01, ***P<0.001; ns: no significant difference.

detailed description

The specific embodiments provided by the present invention will be described in detail below in conjunction with the accompanying drawings.

Example 1: Analysis of the effect of estrogen on the autophagy and proliferation of endometrial cancer cell lines Ishikawa and KLE and the effect on glutamine metabolism

1) Estrogen promotes the proliferation of hormone-sensitive endometrial cancer cell line Ishikawa and inhibits autophagy of Ishikawa, but has no obvious effect on insensitive KLE

Firstly, Western blotting method was used to detect and found that the endometrial cancer cell line Ishikawa expressed estrogen receptor α, but KLE did not express (Ishikawa and KLE cell lines were purchased from Shanghai South Model Animal Center, ER-α and Actin antibodies were purchased from Abcam company) (as shown in Figure 1); subsequently estrogen (10nM) and/or fulvestrant (250nM) treat the cell line, CellCounting Kit-8 kit detects respectively different time points (0, 24, 48, 72 hours ) cell proliferation (both estrogen and fulvestrant were purchased from MedChem Express, and the Cell Counting Kit-8 kit was purchased from Yeasen). It was found that estrogen could promote the proliferation of Ishikawa cells, but had no significant effect on KLE (Fig. 2). The expressions of autophagy-related proteins LC3B-I/II, Beclin-1 and p62 were detected by Western blot after treatment of endometrial cancer cell lines with estrogen (10 nM) and/or fulvestrant (250 nM) for 72 hours (LC3B-I/ II, Beclin-1, and p62 were all purchased from CST Company), and the cells were collected for autophagy scanning electron microscopy. It was found that estrogen can inhibit autophagy in Ishikawa cells, but had no significant effect on KLE, indicating that estrogen inhibits hormone-sensitive intrauterine Autophagy of membranous cancer cells (as shown in Figure 3-6).

2) Estrogen promotes glutamine metabolism in hormone-sensitive endometrial cancer cell line Ishikawa, but has no significant effect on insensitive KLE

Real-time fluorescence quantitative reverse transcription polymerase chain reaction (real time qRT-PCR) detected that the transcript level of glutaminase was significantly increased after estrogen treatment of Ishikawa, but KLE had no significant change (real time qRT-PCR kit was purchased from TaKaRa Company, primers Synthesized by Shenggong Company) (as shown in Figure 7). At the same time, the Elisa method was used to detect the concentration of glutamine in the cell culture supernatant, and it was found that estrogen promoted the glutamine metabolism of Ishikawa, but had no significant effect on KLE (glutamine Elisa kit was purchased from R&D company) (as shown in Figure 8) . At the same time, Western blotting and laser confocal methods were used to detect the expression of glutaminase in the cells, and it was found that estrogen stimulation promoted the expression of glutaminase in Ishikawa cells, but had no significant effect on KLE (glutaminase antibody was purchased from Abcam company) (as shown in Figure 9-10). It shows that estrogen can promote the expression of glutaminase in hormone-sensitive endometrial cancer cell line Ishikawa, and then promote glutamine metabolism, but has no obvious effect on insensitive KLE.

Example 2: In vivo and in vitro experiments prove that glutaminase inhibitor CB-839 effectively inhibits glutamine metabolism in endometrial cancer cells

1) CB-839 inhibits glutamine metabolism in endometrial cancer cell lines Ishikawa and KLE

Concentration gradient (0, 0.02, 0.2, 2, 20, 200mM) glutamine was used to treat Ishikawa and KLE, CellCounting Kit-8 experiments showed that glutamine promoted the proliferation of endometrial cancer cells (Figure 11). The glutaminase inhibitor CB-839 (1 μmol/L) can inhibit the metabolism of glutamine in Ishikawa and KLE (Figure 12). Western blotting and autophagy electron microscopy were used to detect the autophagy level of endometrial cancer cells, and it was found that CB-839 can promote autophagy of endometrial cancer cells (as shown in Figure 13-16). It shows that CB-839, a glutaminase inhibitor, promotes the autophagy of endometrial cancer cells and inhibits the proliferation of endometrial cancer cells.

2) Oral administration of CB-839 can effectively inhibit the growth of subcutaneous endometrial carcinoma xenografts in nude mice

Flow cytometry technology detects oral CB-839 (oral dosage is 200mg/kg, every day twice, and the carrier is that the concentration that contains 25% hydroxypropyl-B-cyclodextrin is 10mmol/L sodium citrate solution, pH=2 , hydroxypropyl-B-cyclodextrin and sodium citrate were both purchased from Sigma) the expression level and average fluorescence density of subcutaneous endometrial cancer tumor cell proliferation-related protein ki67 in nude mice were significantly lower than those of mice taking only the drug carrier group (ki67 antibody was purchased from Biolegend), indicating that oral administration of CB-839 can inhibit the proliferation of endometrial cancer cells (as shown in Figure 17). The growth curve of the subcutaneously implanted tumor in nude mice, the appearance of the tumor body, and the monitoring of the weight of the tumor indicate that oral administration of CB-839 can effectively inhibit the growth of the subcutaneously implanted tumor of endometrial cancer (as shown in Figure 18-20).

Example 3: In vivo and in vitro experiments prove that the glutaminase inhibitor CB-839 effectively inhibits the role of estrogen in promoting glutamine metabolism

1) CB-839 effectively inhibits the promoting effect of estrogen on glutamine metabolism in endometrial cancer cell lines

Cell Counting Kit-8 was used to detect cell proliferation in the untreated group, the estrogen stimulation group, and the estrogen and CB-839 direct stimulation group, and it was found that CB-839 could inhibit the proliferation-promoting effect of estrogen (as shown in Figure 21). Western blotting and autophagy scanning electron microscopy were used to detect the differences in the levels of autophagy among the three groups, and it was found that CB-839 could effectively reverse the inhibition of autophagy caused by estrogen (as shown in Figure 22-25). It shows that CB-839 can effectively inhibit the proliferation of endometrial cancer cells induced by estrogen, and reverse its inhibition of autophagy.

2) Oral administration of CB-839 effectively reversed the promoting effect of estrogen on the growth of endometrial cancer subcutaneous xenografts in nude mice

Flow cytometry analysis technique detects the ratio of ki67-positive cells of endometrial cancer subcutaneous transplanted tumor cells in nude mice in the untreated group, the estrogen application group, the oral CB-839 group, and the simultaneous application of estrogen and CB-839 groups (as shown in Figure 26 -27). Tumor growth detection, subcutaneous tumor appearance and tumor weight detection showed that oral administration of CB-839 could effectively reverse the promoting effect of estrogen on the growth of endometrial cancer subcutaneous transplanted tumors in nude mice (as shown in Figures 28-30).

The results of in vitro and in vivo tests of the present invention confirm that the glutaminase inhibitor CB-839 can effectively inhibit the promoting effect of estrogen on hormone-sensitive endometrial cancer cells, inhibit the proliferation of endometrial cancer cells and promote their apoptosis, Conducive to limiting the growth of endometrial cancer tumors. Oral administration of CB-839 improves abnormal growth of estrogen-induced hormone-sensitive endometrial cancer.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, some improvements and supplements can also be made, and these improvements and supplements should also be considered Be the protection scope of the present invention.

Claims (3)

1. applications of the glutaminase inhibitor C B-839 in the medicine for preparing treatment estrogen sensitive type carcinoma of endometrium.

2. applications of the glutaminase inhibitor C B-839 in preparation of preparation, it is characterised in that described reagent is used for：

A) the external metabolism for suppressing endometrial carcinoma cell GLN,

It is b) external to promote endometrial carcinoma cell autophagy,

It is c) external to suppress endometrial carcinoma cell propagation,

D) the rush endometrial carcinoma cell proliferation function that external suppression estrogen causes, and/or

E) the external suppression endometrial carcinoma cell autophagocytosis for reversing estrogen to cause.

3. application according to claim 2, it is characterised in that described endometrial carcinoma cell is Ishikawa or KLE Cell.