CN110914688A - Biomarker for predicting gemcitabine drug sensitivity and application thereof - Google Patents
Biomarker for predicting gemcitabine drug sensitivity and application thereof Download PDFInfo
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Abstract
The invention provides the use of AKR1C1, AKR1C2, or AKR1C3 as biomarkers for predicting drug sensitivity of gemcitabine, or a pharmaceutically acceptable salt thereof, in the treatment of cancer. The cancer is colon cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, melanoma, hepatocarcinoma, breast cancer, and cervical cancer. The technical scheme of the invention is utilized to guide clinical medication, which not only can avoid blindness of medicine selection and delay of patient's state of illness due to improper medicine selection, but also can monitor the medicine treatment effect in the treatment process, and realize the rationality of medication and the purpose of individual treatment.
Description
The invention relates to the fields of molecular biology and medicine, in particular to a biomarker for predicting gemcitabine drug sensitivity and application thereof.
Gemcitabine is a commonly used anti-tumor chemotherapeutic drug in clinical practice. Gemcitabine hydrochloride is suitable for treating inoperable advanced or metastatic pancreatic cancer and locally advanced or metastatic non-small cell lung cancer, and is combined with paclitaxel to treat breast cancer which recurs after adjuvant/neoadjuvant chemotherapy, cannot be resected, locally recurs or metastasizes. Even with the current advent of targeted and novel immunotherapeutic drugs, gemcitabine remains in widespread clinical use, particularly in the pancreatic cancer field, as a standard therapeutic drug. However, during clinical treatment, there are major differences in the sensitivity or efficacy of gemcitabine among different patients and problems with primary and acquired resistance. Therefore, the effective marker of gemcitabine drug sensitivity is searched for guiding the medication, which not only can avoid delaying the treatment of patients due to improper drug selection, but also can monitor the treatment effect of the drugs in the treatment process, thereby realizing the purposes of medication rationality and individualized treatment.
At present, gemcitabine resistance is considered to be mainly related to uptake and transport process disorder, intracytoplasmic activation, catabolic enzyme abnormality and DNA repair, and research contents include genes or proteins such as hENT1 (human equilibrium nucleoside transporter 1), RRM1 (ribonucleotide reductase M1), RRM2 (ribonucleotide reductase M2), dCK (deoxycytidine kinase) and the like.
There are also many studies on these genes or proteins, but the results vary from study to study. Researchers such as RolandInderson (World J gastroenterol.2014; 20: 8482- & 8490) performed a systematic review of 10 clinical studies involving 855 patients divided into high and low hENT1 groups, with the results showing that overall survival was significantly longer in high expressing hENT patients than in low expressing ones. However, in a randomized phase III clinical study, CONKO-001 clinical trial (Eur J cancer. 2015; 51: 1546-1554), in tissue samples from patients receiving adjuvant gemcitabine chemotherapy, hENT1 expression was detected by immunohistochemistry using SP120 rabbit mab, which confirmed that hENT1 had no predictive effect. Yet another clinical study (NCT01124786, J Clin Oncol.2013; 31:4453-4461) also showed that hENT1 had no predictive effect on gemcitabine sensitivity. Fujit et al (Neoplasia.2010; 12: 807-817) examined mRNA from hENT1, dCK, RRM1, RRM2 in 70 resected PDAC patients, 40 of whom received gemcitabine-based adjuvant chemotherapy. The results of the study showed a longer disease progression free survival in patients with high expression of dCK and low RRM 2. Another study by Maretechal et al (gastroenterology.2012; 143: 664-674. e1-6) showed that patients with high expression of hENT1 and dCK could be predicted to have longer survival when receiving gemcitabine-assisted therapy. The reasons for these large differences in study results include different detection methods used by researchers, complex patient medication, tumor heterogeneity, small sample size, etc. In addition to this, it is possible that no more accurate prediction index is found.
Disclosure of Invention
Aiming at the problems that different patients have large differences of sensitivity or effectiveness to gemcitabine at present, primary and acquired drug resistance problems exist and uncertainty of gemcitabine sensitivity prediction indexes, the invention tries to find out new and more accurate gemcitabine sensitivity prediction indexes.
There are 15 families in the aldo. keto reductase (AKR) superfamily, with 15 members of the human AKR. Most AKR members catalyze simple redox reactions with NADP, a cofactor. They act on a wide range of substrates, including sugars, fatty aldehydes, steroid hormones, prostaglandins, carcinogens, and the like. Thus, members of the AKR superfamily may be enzymes that play an important role in vital activities such as hormone synthesis, drug metabolism, inflammatory responses, carcinogen detoxification, etc. Research shows that the human AKR superfamily members have relevance with various tumorigenesis and development, and the activity is closely related with the tumor progression and the treatment effect. There is increasing evidence that some members of the AKR superfamily are associated with tumor resistance (International Oncology.2013; 40(1): 43-46). The AKRlB10 becomes an early diagnosis marker of non-small cell lung cancer related to smoking, especially squamous cell lung cancer, and the function characteristics of AKR1C 1-3 in the tolerance process of various chemotherapeutics of lung cancer make the marker become an important factor for prognosis prediction, and also provides an effective function target for improving the drug sensitivity of lung cancer. However, the relationship between the AKR family and gemcitabine resistance has never been studied or reported.
The invention discovers that members of an aldo.keto reductase (AKR) family, namely AKR1C1, AKR1C2 and AKR1C3 families, are related to gemcitabine resistance for the first time. The invention researches the correlation between gemcitabine drug sensitivity and AKR1C1-C3 expression in 38 nude mouse tumor transplantation tumor models. The research results show that in 15 models with high AKR1C1-C3 expression, 13 model results show that gemcitabine has poor efficacy and shows insensitivity; of the 23 models with low AKR1C1-C3 expression, 19 models showed good gemcitabine effect and sensitivity.
Accordingly, it is an object of the present invention to provide biomarkers AKR1C1, AKR1C2, AKR1C3 and uses thereof for predicting drug sensitivity of gemcitabine or a pharmaceutically acceptable salt thereof when treating cancer.
In one aspect, the invention provides the use of AKR1C1, AKR1C2, or AKR1C3 as a biomarker for predicting drug sensitivity of gemcitabine, or a pharmaceutically acceptable salt thereof, when used to treat cancer.
According to an embodiment of the invention, the pharmaceutically acceptable salt comprises an acid addition salt of gemcitabine with an acid, for example an acid addition salt of gemcitabine with an acid selected from the group consisting of: inorganic acids such as hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, or nitric acid; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, succinic acid, malic acid, adipic acid, succinic acid, Mandelic acid, ascorbic acid, glucoheptylic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.
As an example, the pharmaceutically acceptable salt is gemcitabine hydrochloride.
According to an embodiment of the present invention, the cancer is colon cancer, lung cancer, stomach cancer, pancreatic cancer, ovarian cancer, melanoma, liver cancer, breast cancer, cervical cancer.
The lung cancer comprises non-small cell lung cancer, the non-small cell lung cancer comprises squamous carcinoma, adenocarcinoma, adenosquamous carcinoma, large cell carcinoma and the like, and the non-small cell lung cancer is preferably lung squamous carcinoma.
In another aspect, the present invention provides a method for predicting drug susceptibility of a drug comprising gemcitabine or a pharmaceutically acceptable salt thereof for the treatment of cancer.
According to an embodiment of the invention, the method comprises applying AKR1C1, AKR1C2 or AKR1C3 as biomarker.
The method may further comprise detecting the expression level of AKR1C1, AKR1C2, or AKR1C3 in the patient in vivo or ex vivo sample.
According to embodiments of the present invention, a therapeutically effective amount of gemcitabine, or a pharmaceutically acceptable salt thereof, may be administered as a single agent or in combination with one or more other agents, wherein the combination does not cause unacceptable adverse effects. Suitable active agents in such combinations include conventional agents suitable for use in chemotherapy or targeted therapy, such as alkylating agents, platinum compounds, DNA modifiers, topoisomerase inhibitors, microtubule modifiers, antimetabolites, anticancer antibiotics, hormones/antagonists, aromatase inhibitors, small molecule kinase inhibitors, photosensitizers, antibodies, cytokines, drug conjugates, vaccines or other drugs, and the like.
The pharmaceutically acceptable carrier may be one that is relatively non-toxic and non-injurious to a patient at concentrations consistent with effective activity of the active ingredient, such that any side effects caused by the carrier do not destroy the beneficial effects of the active ingredient. A pharmaceutically effective amount of a compound or a pharmaceutically acceptable salt thereof is preferably an amount that results in, or affects, the particular condition being treated. Gemcitabine, or a pharmaceutically acceptable salt thereof, may be administered with pharmaceutically acceptable carriers well known in the art in any effective conventional dosage unit form, including immediate release, sustained release and timed release formulations, in the following manner: oral, parenteral, topical, nasal, ocular, sublingual, rectal, vaginal, and the like.
In another aspect, the present invention provides a kit for predicting drug susceptibility of a drug containing gemcitabine or a pharmaceutically acceptable salt thereof in treating cancer, the kit comprising:
and (b) a reagent for detecting the expression level of at least one of AKR1C1, AKR1C2 and AKR1C3 in the individual sample.
The individual sample is selected from human cancer tissue, specifically, colon cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, melanoma, liver cancer, breast cancer, and cervical cancer.
The invention has the beneficial effects that: the Aldehyde Ketone Reductase (AKR) family members AKR1C1, AKR1C2 or AKR1C3 are used as biomarkers for predicting drug sensitivity of gemcitabine in cancer treatment to guide clinical medication, so that the blindness of drug selection can be avoided, the condition of a patient can be prevented from being delayed due to improper drug selection, the therapeutic effect of the drug can be monitored in the treatment process, and the rationality of medication and the purpose of individual treatment can be realized. The expression test results of clinical cancer cases AKR1C1-C3 further verify the high correlation between the expression of AKR1C1-C3 and the curative effect of gemcitabine, and illustrate the rationality, feasibility and high effectiveness of using AKR1C1, AKR1C2 or AKR1C3 to predict the curative effect of gemcitabine on cancer.
FIG. 1 analysis of the expression levels of AKR1C1-C3 in different cells GAPDH or β -tubulin as an internal reference the higher the grayscale value of the bands, the higher the expression level of the corresponding protein.
FIG. 2 anti-tumor effect of gemcitabine in different colon cancer nude mouse graft tumor models.
FIG. 3 anti-tumor effect of gemcitabine in different nude mice transplantation tumor models of lung cancer.
FIG. 4 shows the antitumor effect of gemcitabine in different nude mice transplantation tumor models with gastric cancer.
FIG. 5 antitumor effect of gemcitabine in different nude mice transplantation tumor models of pancreatic cancer.
FIG. 6 antitumor effect of gemcitabine in different ovarian cancer nude mouse graft tumor models.
FIG. 7 antitumor effect of gemcitabine in HepG2 nude mouse transplanted tumor model.
Figure 8 antitumor effect of gemcitabine in HeLa cervical cancer graft tumor model.
FIG. 9 antitumor effect of gemcitabine in MDA-MB-435 melanoma nude mouse graft tumor model.
The following further illustrates the invention and should not be construed as limiting the spirit and scope of the invention.
Experiment one: western immunoblotting (WB, Western blot)
The experimental principle is as follows:
a protein sample separated by polyacrylamide gel electrophoresis (PAGE) is transferred and combined on a solid phase carrier (such as a nitrocellulose membrane, a difluorinated resin membrane and the like), the protein or polypeptide on the solid phase carrier is used as an antigen, a corresponding antibody can be identified and combined with the antigen, then the antibody reacts with an enzyme or isotope labeled second antibody, and the expression quantity of the target protein is detected by substrate color development or autoradiography.
Purpose of the experiment:
detecting the expression of the AKR1C1-C3 proteins in different cells.
Experimental materials:
cell: human colon cancer cells CW-2, LoVo, HCT116, SW620, COLO205, HT-29, RKO, DLD-1, NCI-H716, SW1116, SW 948; human lung cancer cells NCI-H292, A549, NCI-H460, NCI-H1650, HCC827, NCI-H1299, NCI-H1975, SK-MES-1, NCI-H1437, NCI-H1944; human gastric cancer cells BGC-823, HGC-27, SGC-7901, KATO III, NCI-N87, SNU-1; human pancreatic cancer cells AspC-1, BxPC-3, PANC-1, Capan-2, CFPAC-1, MIA PaCa-2, HPAF-II; human breast cancer cells BT-474, SK-BR-3, MCF-7, T-47D, MDA-MB-231, MDA-MB-468; human ovarian cancer cells SK-OV-3, NIH OVCAR-3, A2780; human hepatoma cell BEL-7402, Hep G2; human cervical cancer cell HeLa; human melanoma cells MDA-MB-435 were purchased from the cell bank of the Committee for type culture Collection of the national academy of sciences and cultured under the cell culture conditions provided on this website.
Primary antibody AKR1C1 murine monoclonal antibody (MAB6529) was purchased from R & D Systems, AKR1C2 rabbit polyclonal antibody (13035) was purchased from Cell Signaling Technology, AKR1C3 murine monoclonal antibody (MAB7678) was purchased from R & D, GAPDH rabbit monoclonal antibody (2118) was purchased from Cell Signaling Technology, HRP- β -tubulin murine monoclonal antibody (ab012) was purchased from a consortium.
Secondary antibody: HRP-labeled goat anti-rabbit IgG and HRP-labeled horse anti-mouse IgG were both purchased from Cell Signaling Technology.
The experimental method comprises the following steps:
cells at 25cm2Culturing in a culture bottle, collecting cells when the coverage rate is 80-90%, adding 200-l, 1% NP-40, 1% SDS,1 XProtease inhibitor Cocktail), vortexed, mixed and lysed on ice for 30 min. After centrifugation at 14000rpm and 4 ℃ for 10min, the supernatant was taken and quantified with a BSA protein quantitative detection kit. And 5 × loading buffer is added and boiled.
30 μ g of total protein of each sample was separated by 10% SDS-PAGE electrophoresis, 250mA was transferred to PVDF membrane by electrotransfer, blocked with 5% skimmed milk powder formulated with TBST for 1h at room temperature, incubated overnight at 4 ℃ with primary antibody, AKR1C1 antibody was diluted 1000 times with 5% skimmed milk powder, AKR1C2 antibody was diluted 500 times, AKR1C3 antibody was diluted 2000 times, GAPDH or β -tubulin antibody was diluted 10000 times, washed thoroughly with TBST, incubated with secondary antibody (5% BSA 5000 times) for 1h at room temperature, washed thoroughly with TBST again, ECL chemiluminescent substrate was added for color development, exposed, the membrane for detection of β -tubulin was incubated with primary antibody, incubated without secondary antibody, washed and directly with ECL chemiluminescent substrate for exposure, GAPDH or β -tubulin was used as an internal reference.
The experimental results are as follows:
the expression amounts of three proteins AKR1C1-C3 in different cells are shown in FIG. 1, and it can be seen from FIG. 1 that:
in human colon cancer cells, AKR1C1-C3 in COLO205 cells are highly expressed; the expression levels of AKR1C1 and AKR1C2 in NCI-H716 cells were moderate to high, and the expression levels of AKR1C3 were high; three proteins in HCT116, RKO and DLD-1 cells are all low expressed; AKR1C1 and AKR1C2 are low in CW-2, LoVo, SW620, HT-29, SW1116 and SW948 cells, and AKR1C3 is medium or high in expression.
In human lung cancer cells, AKR1C1-C3 in A549 cells, NCI-H460 cells, SK-MES-1 cells, NCI-H1437 cells and NCI-H1944 cells are highly expressed; the expression of AKR1C1-C3 in NCI-H292, NCI-H1650, NCI-H1299 and NCI-H1975 cells is low; in HCC827 cells, AKR1C1-C3 were all low expressed.
In human gastric cancer cells, AKR1C1-C3 in SGC-7901 cells is highly expressed; AKR1C1-C3 in HGC-27 and SNU-1 are low in expression; the expression level of AKR1C1 in BGC-823 cells is moderate, the expression level of AKR1C2 is low to moderate, and the expression level of AKR1C3 is high; in KATOIII and NCI-N87 cells, AKR1C1 and AKR1C2 proteins were low expressed, and AKR1C3 was medium expressed.
In human pancreatic cancer cells, AKR1C1-C3 in AsPC-1 and BxPC-3 cells are highly expressed; AKR1C1-C3 in PANC-1 and Capan-2 are low in expression; in CFPAC-1 cells, AKR1C1 and AKR1C2 are expressed at low to moderate levels, and AKR1C3 is expressed at high levels; high expression in AKR1C1 and low expression in AKR1C2 and AKR1C3 in MIA PaCa-2 cells; in HPAF-II cells, AKR1C1 was expressed at a low level, AKR1C2 was expressed at a low to moderate level, and AKR1C3 was expressed at a high level.
In human breast cancer cells, AKR1C1-C3 in BT-474, MCF-7, T-47D, MDA-MB-231 and MCF7 cells are all low expressed; AKR1C1 and AKR1C2 in SK-BR-3 cells are low in expression, and AKR1C3 is medium in expression; AKR1C1 and AKR1C2 are highly expressed in MDA-MB-468 cells, and AKR1C3 is moderately expressed.
In human ovarian cancer cells, NIH, namely, in OVCAR-3 and A2780 cells, AKR1C1-C3 are low in expression; AKR1C1-C3 in SK-OV-3 cells are highly expressed.
In human liver cancer cells, AKR1C1-C3 in BEL-7402 cells are low expressed; in Hep G2 cells, AKR1C1 and AKR1C2 were expressed at low to moderate levels, and AKR1C3 was expressed at high levels.
In human cervical carcinoma cells HeLa, both AKR1C1 and AKR1C2 are low in expression, and AKR1C3 is high in expression.
In human melanoma cells MDA-MB-435, AKR1C1-C3 are all low in expression.
Experiment two: drug effect test of transplanted tumor in nude mouse
Purpose of the experiment: the effect of gemcitabine on transplanted tumors of nude mice with different tumors was studied.
Experimental materials:
gemcitabine hydrochloride was purchased from Melphalan, matrigel was purchased from Corning Inc. of America (cat # 354262), and BALB/c nude mice were purchased from Shanghai Spill-Bikay laboratory animals Co.
The experimental method comprises the following steps: colon cancer cells CW-2, LoVo, HCT116, SW620, COLO205, HT-29, RKO, DLD-1, NCI-H716; human lung cancer cells NCI-H292, A549, NCI-H460, NCI-H1650, HCC827, NCI-H1299, NCI-H1975, SK-MES-1, NCI-H1944, NCI-H1437; human gastric cancer cells BGC-823, HGC-27, SGC-7901, NCI-N87 and SNU-1; human pancreatic cancer cells AsPC-1, BxPC-3, PANC-1, Capan-2, CPFAC-1, MIA PaCa-2 and HPAF-II; human breast cancer cells MDA-MB-231; human ovarian cancer cells SK-OV-3, A2780; human liver cancer cells BEL-7402, HepG 2; human cervical cancer cell HeLa; after in vitro culture of human melanoma cells MDA-MB-435, collecting cell suspension in exponential growth phase to appropriate concentration, mixing with matrigel 1:1, inoculating with BALB/c nude mouse subcutaneous tumor, establishing various transplanted tumor models, and dividing into 2 groups after the tumor grows to 100-200 mm3, namely a control group and a gemcitabine administration group, wherein each group comprises 6 animals. The gemcitabine hydrochloride is administrated by intraperitoneal injection at the dose of 120mg/kg, and is administrated on the 1 st day and the 4 th day of a week. Tumor major axis a (mm) and minor axis b (mm) were measured 2 times per week, and tumor volume (V) was calculated according to the following formula: v-1/2 × a × b2(mm 3); calculating Relative Tumor Volume (RTV) according to the tumor volume result, wherein the calculation formula is as follows: RTV is Vt/V0. Where V0 is the tumor volume measured at the time of caged administration (i.e., d0) and Vt is the tumor volume at each measurement. The evaluation index of the antitumor activity is relative tumor proliferation rate T/C (%), and the calculation formula is as follows: T/C% ═ TRTV/CRTV 100%; wherein TRTV: treatment group mean RTV; CRTV: vehicle control group mean RTV. And (4) counting by using an Excel table and carrying out T.test test.
The experimental results are as follows:
gemcitabine shows differences in its potency among different models of transplants.
The antitumor effects of gemcitabine in different nude mouse transplantable tumor models vary widely under the same dose and regimen. In 9 colon cancer transplantable tumor models (see figure 2), gemcitabine had a significant inhibitory effect on the growth of HCT-116, HT-29, CW-2, LoVo, RKO, DLD-1 transplantable tumors, with T/C% < 20%; however, in COLO205 and SW620 transplantation tumor models, the inhibition effect of gemcitabine is poor, and the T/C% is more than 40%; in the NCI-H716 model, gemcitabine inhibited intermediately with a T/C% of about 38%.
Similarly, gemcitabine has significant inhibitory effects on NCI-H1650, NCI-H1975 and NCI-H1299 nude mouse transplantants with T/C < 20% in a lung cancer transplant model (see FIG. 3); and poor inhibitory effect on A549, NCI-H460, NCI-H1437 and NCI-H1944 transplantable tumors (see FIG. 3), T/C > 40%.
In the gastric cancer model (see FIG. 4), gemcitabine has a significant inhibitory effect on the growth of NCI-N87, HGC-27 and SNU-1 transplantable tumors, T/C < 20%; but has poor inhibitory effect on BGC-823 and SGC-7901 transplantable tumors, with T/C > 50%.
In a pancreatic cancer model (see FIG. 5), gemcitabine has a significant inhibitory effect on Capan-2, PANC-1, CFPAC-1 transplantable tumors, T/C < 20%; has poor inhibition effect on BxPC-3 and AsPC-1 transplantable tumor, and T/C is more than 40 percent.
Similar phenomena were observed in the ovarian cancer model (see FIG. 6), with gemcitabine having a greater differential inhibitory effect on A2780 and SK-OV-3 transplants, with a significant inhibitory effect on A2780 and a lesser effect on the SK-OV-3 model. Gemcitabine inhibited poorly in the liver cancer model (see fig. 7) HepG2 transplantable tumors, with T/C > 50%.
In the cervical cancer model (see FIG. 8), gemcitabine inhibited poorly against HeLa transplantable tumor grafts, with T/C > 50%.
Gemcitabine also showed strong antitumor activity in the MDA-MB-435 melanoma model (see FIG. 9), T/C < 20%.
The research results of the first experiment and the second experiment show that: a strong correlation exists between gemcitabine sensitivity and AKR1C1-C3 expression, specifically, gemcitabine has poor efficacy and shows insensitivity in a model with high AKR1C1-C3 expression; in the model with low AKR1C1-C3 expression, gemcitabine has good efficacy and shows sensitivity. Therefore, AKR1C1-C3 can be used as a biomarker for predicting drug sensitivity of gemcitabine or a pharmaceutically acceptable salt thereof in cancer treatment.
Experiment three: detection of the expression of AKR1C1-C3 protein in Gemcitabine-treated Lung squamous cell carcinoma cases
Purpose of the experiment: the correlation between the expression level of AKR1C1-C3 and the gemcitabine curative effect is verified retrospectively by clinical lung squamous carcinoma cases.
Experimental materials:
paraffin-embedded pathological tissues of 36 patients with squamous cell lung carcinoma who had used gemcitabine were provided by the tumor hospital in Zhejiang province. AKR1C1 Rabbit polyclonal antibody (ab192785) was purchased from abcam, and AKR1C2 antibody (AM05295PU-N) was purchased from Origene; the AKR1C3 antibody (ab49680) was purchased from abcam; BOND III full-automatic dyeing machine was purchased from Leica (Germany).
The experimental method comprises the following steps:
and (3) slicing the paraffin sample to be about 4 mu m in thickness, drying the paraffin sample in an oven, and placing the paraffin sample in a BOND III full-automatic dyeing machine for immunohistochemical detection. The primary antibody is diluted at a ratio of 1:200, the incubation time is 15min, and the incubation time of the BOND Polymer Refine is 8 min. Finally, the tissue sections are scored according to the staining degree under a light microscope, and the expression level of AKR1C1-C3 in different samples is judged.
The experimental results are as follows:
the expression levels of AKR1C1-C3 in squamous cell lung carcinoma were correlated with the therapeutic effect of gemcitabine (Table 1)
Of the 36 cases of lung squamous carcinoma with gemcitabine, 15 cases were evaluated as Partial Response (PR), 15 cases were evaluated as Stable Disease (SD), and 6 cases were evaluated as Progressive Disease (PD), so the objective effective rate was 41.7% (15/36).
Of the 36 patients, 27 detected high expression of AKR1C1-C3, and the efficacy evaluations were: 8 cases of PR, 13 cases of SD and 6 cases of PD, therefore, the objective effective rate is 29.6 percent (8/27). In 9 cases, the low expression of AKR1C1-C3 was detected, and the curative effect evaluation was: since 7 cases of PR and 2 cases of SD, the objective effective rate was 77.8% (7/9).
And (3) displaying a statistical result: among squamous cell lung carcinoma, patients with low expression of AKR1C1-C3 showed better therapeutic effect after gemcitabine (77.8% and 29.6% for objective effective rate, respectively). The objective effective rate of 36 patients is 41.7%, while the objective effective rate of AKR1C1-C3 low-expression patients is 77.8%, so that the effective rate of gemcitabine can be improved by screening the lung squamous carcinoma patients with low-expression AKR1C1-C3 by detecting the expression quantity of AKR1C 1-C3. In conclusion, retrospective clinical case studies show that AKR1C1-C3 can be used as a biomarker of clinical efficacy of gemcitabine and can be used for predicting the efficacy of gemcitabine.
TABLE 1 correlation analysis of AKR1C1-C3 expression level in squamous cell lung carcinoma with gemcitabine curative effect
Claims (10)
- Use of AKR1C1, AKR1C2 or AKR1C3 as a biomarker for predicting drug sensitivity of gemcitabine or a pharmaceutically acceptable salt thereof in the treatment of cancer.
- The use of claim 1, wherein the pharmaceutically acceptable salt is an acid addition salt of gemcitabine with an acid selected from the group consisting of: hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, or nitric acid; formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, mandelic acid, ascorbic acid, glucoheptylic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.
- The use of claim 1 or 2, wherein the pharmaceutically acceptable salt is gemcitabine hydrochloride.
- The use according to any one of claims 1 to 3, wherein the cancer is colon cancer, lung cancer, stomach cancer, pancreatic cancer, ovarian cancer, melanoma, liver cancer, breast cancer, cervical cancer, preferably the lung cancer is non-small cell lung cancer, more preferably squamous cancer, adenocarcinoma, adenosquamous carcinoma, large cell carcinoma.
- A method of predicting drug sensitivity in the treatment of cancer with a drug comprising gemcitabine or a pharmaceutically acceptable salt thereof, wherein the method comprises administering AKR1C1, AKR1C2, or AKR1C3 as a biomarker.
- A method for predicting drug sensitivity in the treatment of cancer with a drug comprising gemcitabine or a pharmaceutically acceptable salt thereof, wherein the method comprises detecting the expression level of AKR1C1, AKR1C2, or AKR1C3 in a sample of a patient in vivo or ex vivo.
- The method of claim 5 or 6, wherein the pharmaceutically acceptable salt is an acid addition salt of gemcitabine with an acid selected from the group consisting of: hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, or nitric acid; formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, mandelic acid, ascorbic acid, glucoheptylic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.
- The method of claim 5 or 6, wherein the cancer is colon cancer, lung cancer, stomach cancer, pancreatic cancer, ovarian cancer, melanoma, liver cancer, breast cancer, cervical cancer, preferably the lung cancer is non-small cell lung cancer, more preferably squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma.
- A kit for predicting drug susceptibility of a drug containing gemcitabine or a pharmaceutically acceptable salt thereof for the treatment of cancer, the kit comprising:and (b) a reagent for detecting the expression level of at least one of AKR1C1, AKR1C2 and AKR1C3 in the individual sample.
- The kit according to claim 9, wherein the individual sample is selected from human cancer cell tissue, in particular, the cancer cell tissue is selected from colon cancer, lung cancer, stomach cancer, pancreatic cancer, ovarian cancer, melanoma, liver cancer, breast cancer, cervical cancer cells, preferably non-small cell lung cancer cells, more preferably squamous cancer, adenocarcinoma, adenosquamous cancer, large cell carcinoma cells.
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| PCT/CN2018/100371 WO2019034042A1 (en) | 2017-08-14 | 2018-08-14 | Biomarker for predicting drug sensitivity to gemcitabine and use thereof |
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| BO-HYUN CHOI等: "Shadows of NRF2 in cancer: Resistance to chemotherapy" * |
| MASATSUGU UEDA等: "Infection of human papillomavirus and overexpression of dihydrodiol dehydrogenase in uterine cervical cancer" * |
| 吴媛媛;: "Keap1/Nrf2信号通路对人肺鳞癌细胞生长、转移和耐药的影响" * |
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