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Application of TRPC5 as drug target in reversing multi-drug resistance

A multi-drug resistance and drug technology, which can be used in anti-tumor drugs, drug combinations, medical preparations containing active ingredients, etc., and can solve problems such as unseen applications

Active Publication Date: 2012-12-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, there is no application of TRPC5 as a drug target in reversing tumor multidrug resistance at home and abroad

Method used

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  • Application of TRPC5 as drug target in reversing multi-drug resistance
  • Application of TRPC5 as drug target in reversing multi-drug resistance
  • Application of TRPC5 as drug target in reversing multi-drug resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Expression level of TRPC5 in multidrug resistant tumor cells and detection of calcium ion permeation activity

[0032] experimental method:

[0033] Collect 1.0x10 each of MCF-7 / WT and MCF-7 / ADM 5 The differences in the expression levels of P-gp and TRPC5 in MCF-7 / WT and MCF-7 / ADM were detected by Western blot.

[0034] HEKA EPC9-9 patch clamp system was used to detect MCF-7 / WT and MCF-7 / ADM and the whole-cell currents of MCF-7 / WT and MCF-7 / ADM after being treated with various TRPC5 inhibitors. The clamping voltage of the cell is 0mV, the current-voltage relationship is -100mV~+100mV, the interval is 20 mV, and the duration is 500ms. The composition of the electrode inner solution: 130mM Cs-aspartate Cs-aspartate, 2 mM MgCl 2 , 5 mM Na 2 ATP, 5.9 mM CaCl 2 , 10 mM EGTA, 10 mM hydroxyethylpiperazine ethylsulfuric acid Hepes (pH7.2); the electrode external fluid is a hypotonic solution with an osmotic pressure of 210mOsm, and its composition is: 65mM Na + -...

Embodiment 2

[0040] Example 2 Detection of relationship between TRPC5 and P-gp expression levels

[0041] MCF-7 / ADM was seeded in a six-well plate at a density of 300,000 cells per well, and after 12 hours of culture, the dominant inhibitory plasmid pCDNA6-TRPC5-DN of TRPC5 was transfected (4 μg / well, 8 μg / well ), with pCDNA6 empty plasmid as the control group, and TRPC5 siRNA siC5 (100pmol / well) (the intentional strand is: CCA AUG GAC UGA ACC AGC UUU ACU U), with negative siRNA as the control group; The cells were routinely cultured, and the cells in each well were collected after 24 hours. According to the conventional operation method, the expression of P-gp in the cells before and after TRPC5 was inhibited was detected by Western blot. Among them, the primary antibody for P-gp incubation was Anti-P-gp (P-glycoprotein), the primary antibody for incubation of TRPC5 is TRPC5 antibody (ab63151), the primary antibody for internal reference βtubulin is β tubulin antibody (SC-9104), and the s...

Embodiment 3

[0044] Example 3 Reversal effect of TRPC5 inhibitors on multidrug resistance of tumor cells in vitro

[0045] experimental method:

[0046] MCF-7 / ADM and MCF-7 / WT were inoculated on 25cm 2 When the MCF-7 / ADM confluence was 80%, the dominant suppressor plasmid pCDNA6-TRPC5-DN (16 μg / flask) of TRPC5 was transfected respectively, and the pCDNA6 empty plasmid was used as the control group, and the SiRNA siC5 of TRPC5 ( 200pmol / bottle), negative siRNA was used as the control group; the cells were cultured routinely, and after 24 hours, the above-treated MCF-7 / ADM and untreated MCF-7 / WT were collected respectively, and 7000 cells per well The density was inoculated in a 96-well plate. After 24 hours, doxorubicin was added according to the concentration gradient. After culturing for 48 hours, the drug sensitivity of the cells was detected by the MTT method.

[0047] Experimental results:

[0048] See the experimental results image 3 . Depend on image 3 -A and 3-B, it can be s...

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Abstract

The invention provides a novel purpose of TRPC5, which is an application of TRPC5 as drug target in reversing multi-drug resistance. The drug is an inhibitor for TRPC5, which comprises a pharmacology antagonist 2-APB of TRPC5, a blocking antibody T5E3 of TRPC5, a dominant inhibition plasmid pCDNA6-TRPC5-DN and Lenti-TRPC5-DN of TRPC5 and SiRNAsiC5 of TRPC5, and the multidrug resistance is generated by P-gp mediation. The invention has the key points that the TRPC5 is closely related to a P-gp protein expression of multidrug resistance generated by mediated tumor, and a TRPC5 inhibitor has obvious reversing effect on multidrug resistance of in vitro and in vivo tumor cells, accordingly, the application of TRPC5 as drug target in reversing multi-drug resistance provides the novel target for antineoplastic multidrug resistance medicine, and provides a novel clue for reversing the tumor multidrug resistance.

Description

technical field [0001] The invention relates to a new application of TRPC5, in particular to the application of the transient receptor potential channel TRPC5 as a drug target in reversing tumor multidrug resistance. Background technique [0002] Tumor multidrug resistance (MDR) refers to the development of cross-resistance to multiple anti-tumor drugs with different structures and mechanisms of action after tumor cells are exposed to a certain anti-tumor drug. , thereby greatly reducing the efficacy of anticancer drugs. Over the years, researchers have discovered a variety of MDR production mechanisms, among which, P-glycoprotein (P-glycoprotein, P-gp) encoded by the multidrug resistance gene MDR1 is currently the most studied and most important multidrug resistance-related protein. . The overall expression level of P-gp in tumor tissues is low, but it has a certain level of expression in malignant tumors, especially in drug-resistant tumor tissues, its expression is sign...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K48/00A61K39/395A61K31/69A61P35/00
Inventor 金坚马鑫陈蕴蔡燕飞何冬旭
Owner JIANGNAN UNIV
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