CN109897866A - A method of reversing the drug resistance of liver cancer cells sorafenib - Google Patents
A method of reversing the drug resistance of liver cancer cells sorafenib Download PDFInfo
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- CN109897866A CN109897866A CN201711307093.2A CN201711307093A CN109897866A CN 109897866 A CN109897866 A CN 109897866A CN 201711307093 A CN201711307093 A CN 201711307093A CN 109897866 A CN109897866 A CN 109897866A
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- sorafenib
- liver cancer
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Abstract
The present invention discloses a kind of newfound access that can be had an impact to sorafenib drug resistance-proline synthesis access.Specifically interfered in SMMC-7721 cell using shRNA perturbation technique and falls ALDH18A1, to achieve the purpose that inhibit proline synthesis, it is confirmed by cell viability detection and protein immunoblot experiment detection, the inhibition of proline synthesis can reverse liver cancer cells to the drug resistance of sorafenib, to achieve the purpose that effectively to treat liver cancer.
Description
Technical field
The present invention relates to cytobiology technologies, and in particular to a kind of to be constructed using ALDH18A1-1ShRNA technology
The method of the SMMC-7721 cell line of ALDH18A1 stable low-expression.
Background technique
Liver cancer is the malignant tumour of liver system, and disease incidence accounts for the 55% of global liver cancer incidence, and the death rate occupies world head
Position.Sorafenib (sorafenib) is the targeted drug for being clinically used for treatment liver cancer uniquely ratified by FDA.Sorafenib is
A kind of inhibitor of more kinases target spots, can not only inhibit RAF kinases, can also inhibit vascular endothelial growth factor receptor
(VEGFR) and platelet derived growth factor receptor (PDGFR) receptor.Although being mentioned researches show that Sorafenib is a degree of
The life cycle of clinically liver cancer patient of high three phases, but the medicine response degree in fact in patient is generally lower, and effect is far not
It is fully up to expectations.More and more concerns have been obtained to the drug resistant mechanism of sorafenib in recent years, have caused sorafenib drug resistant
Mechanism is extremely complex.
The micro-environmental hypoxia (Hypoxia) of tumour leads to Sorafenib drug resistance, and tumor hypoxia and traditional chemicotherapy are resistance to
Pharmacological property, metastases and recurrence are highly relevant.There is document report hypoxia inducible factor in the drug resistant liver cancer tissue of sorafenib
The expression of (HIF1 α) is significantly higher than non-drug resistance group, inhibits the expression of HIF2 α and activity that can reverse the cancer cell pair
The drug resistance of sorafenib.Importantly, sorafenib, which is used for a long time, can inhibit the angiogenesis of tumor tissues, increase
Add tumor hypoxia, so as to cause cell to the drug resistance of sorafenib, significantly limits the clinic of sorafenib in this way
Drug effect, but also liver cancer targeting histanoxia becomes more attracting research field.
It is newest research shows that in kidney, lack growth of tumour cell and the survival of L-aminobutanedioic acid or glutamine scarcity
Height relies on the biosynthesis of proline.In Humanmachine tumour, proline synthesis access is inhibited to significantly suppress cancer cell
Proliferation.In addition, newest, researches show that hydroxy-prolines to take part in cancer cell under hypoxia condition as the hinge of metabolism
Survival.
Summary of the invention
The present invention relates to the new method of assisting in treating liver cancer, the first purpose is to illustrate in liver cancer Proline synthesis and face
The drug resistance correlation of the sorafenib as caused by anoxic on bed；The second purpose is explicitly by the biosynthesis for inhibiting proline
To reverse liver cancer to the drug resistance of sorafenib.ShALDH18A1 plasmid is bought from Origene company, and article No. is TL314847.
The experimental results showed that under low oxygen conditions, Proline synthesis promotes the existence of liver cancer cells, increase
The drug resistance of sorafenib.
The present invention discloses a kind of newfound access-proline synthesis that can be had an impact to sorafenib drug resistance
Access.Specifically interfered in SMMC-7721 cell using shRNA perturbation technique and fall ALDH18A1, inhibits proline to close to reach
At purpose, confirm that the inhibition of proline synthesis can reverse by cell viability detection and protein immunoblot experiment detection
Liver cancer cells are to the drug resistance of sorafenib, to achieve the purpose that effectively to treat liver cancer.
Detailed description of the invention
Fig. 1 .SMMC-7721-Scramble and SMMC-7721-shALDH18A1 cell add respectively or are not added 10 μM
Sorafenib is being passed through volumetric concentration 20%O2Or 1%O2After handling 48 hours under gas condition, cell viability detection is carried out.
Fig. 2 .SMMC-7721-Scramble and SMMC-7721-shALDH18A1 cell are respectively in 20%O2Or 1%O2Item
After adding under part or 10 μM of sorafenib processing being not added 24 hours, cell pyrolysis liquid is collected, is tested by western blot, is used
Cell death related protein is detected.
Fig. 3 is the result schematic diagram that embodiment 4 is tested by western blot.
Specific embodiment
Now in conjunction with example, the present invention will be further described.Example is only limitted to illustrate the present invention, rather than to limit of the invention
It is fixed.
It is prepared by the solution of 1 Raf-1, B-Raf and VEGFR-2 multi-kinase inhibitor sorafenib of example.
It is 100mM that Sorafenib, which is prepared into storing liquid concentration with DMSO dissolution,.Sorafenib adds containing 10% fetal calf serum
DMEM culture solution be diluted to 10 μM as working concentration.
Example 2 SMMC-7721-Scramble and SMMC-7721-shALDH18A1 surely turns the building of cell line
ShALDH18A1 plasmid is bought from Origene (TL314847), by shScramble and shALDH18A1 plasmid point
Not and virus package carrier psPAX2, p VSV.G are packaged into viral (mass ratio 4:3:1), then contain 10% fetal calf serum in 4ml
DMEM culture solution in the additional amounts of three kinds of plasmids be followed successively by 12 μ g, 9 μ, 3 μ g, virus infection SMMC-7721 cell, by cell
It passes in 10cm ware, passes on the screening for starting puromycin in second day, beginning concentration is 1ug/ml, a large amount of until no longer occurring
Dead concentration increases to 2ug/ml. and is continued for screening, and after there is clone, picking monoclonal is cultivated.Cell expansion culture
Increase drug concentration 3ug/ul afterwards to continue to screen, until no mortality is further added by drug concentration 4ug/ml, expansion is trained after screening
Silencing efficiency detection is supported and carried out, is frozen after obtaining ideal effect.Its result in SMMC-7721 cell as shown in Figure 1, strike low
The expression of ALDH18A1.
3 proline synthesis of example inhibits the synergy with sorafenib to the inhibiting effect of liver cancer cells.
By SMMC-7721-Scramble and SMMC-7721-shALDH18A1 cell dissociation at individual cells, then carry out
Cell count, by every kind of 3x103A cell is taped against in 96 orifice plates, is divided into 8 each groups, respectively 20%O2SMMC-7721-
Scramble, 20%O2SMMC-7721-Scramble adds sorafenib to handle, 20%O2SMMC-7721-shALDH18A1,
20%O2SMMC-7721-shALDH18A1 adds sorafenib to handle, 1%O2SMMC-7721-Scramble, 1%O2SMMC-
7721-Scramble adds sorafenib to handle, 1%O2SMMC-7721-shALDH18A1,1%O2SMMC-7721-
ShALDH18A1 adds sorafenib to handle, and after sorafenib is handled 24 hours, is carried out using Cell Titer Glo method thin
Born of the same parents' viability examination, result add afterwards as shown in Fig. 2, striking low ALDH18A1 (inhibiting proline synthesis) in SMMC-7721
Sorafenib processing inhibits the proliferation of liver cancer cells under normal oxygen and hypoxia condition.
4 proline synthesis of example inhibits to promote the apoptosis of liver cancer cells.
SMMC-7721-Scramble and SMMC-7721-shALDH18A1 cell is taped against in 6cm ware, 8 each groups are divided into,
Respectively 20%O2SMMC-7721-Scramble, 20%O2SMMC-7721-Scramble adds sorafenib to handle, and 20%
O2SMMC-7721-shALDH18A1,20%O2SMMC-7721-shALDH18A1 adds sorafenib to handle, 1%O2SMMC-
7721-Scramble, 1%O2SMMC-7721-Scramble adds sorafenib to handle, 1%O2SMMC-7721-
ShALDH18A1,1%O2SMMC-7721-shALDH18A1 adds sorafenib to handle, and after sorafenib is handled 24 hours, receives
Cell protein is taken, is detected using western blot experiment.Its result is as shown in figure 3, under normoxic condition, ALDH18A1
Knockout (proline synthesis inhibition) and sorafenib synergistic effect, promote the apoptosis of liver cancer cells；Under low oxygen conditions, single
The apoptosis of liver cancer cells can be promoted by solely knocking out ALDH18A1.
Show that Proline Metabolism access is related to the development of liver cancer by above-mentioned experiment, proline synthesis can make
Sorafenib generates drug resistance and therefore inhibits the synthesis of proline to be able to suppress the growth of tumour cell, make liver cancer cells pair
Sorafenib is sensitive, to achieve the purpose that treat liver cancer.
Claims (3)
1. a kind of method for the drug resistance for reversing liver cancer cells sorafenib, it is characterised in that: in Hepatocellular carcinoma cell line
Interfere the key enzyme-ALDH18A1 in proline synthesis access in cell, by knocking out 18 family member A1 of people's acetaldehyde dehydrogenase
(ALDH18A1) inhibit the synthesis of proline.
2. the method as described in claim 1, which is characterized in that shALDH18A1 plasmid is packaged into virus, then utilizes disease
Poison infects, and is built into SMMC-7721-shALDH18A1 and surely turns cell line.
3. according to the method described in claim 2, it is characterized in that, falling ALDH18A1 by interference to inhibit the conjunction of proline
At the drug resistance of liver cancer cells sorafenib can be reversed.
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| CN201711307093.2A CN109897866A (en) | 2017-12-11 | 2017-12-11 | A method of reversing the drug resistance of liver cancer cells sorafenib |
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| CN201711307093.2A CN109897866A (en) | 2017-12-11 | 2017-12-11 | A method of reversing the drug resistance of liver cancer cells sorafenib |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101999002A (en) * | 2008-02-04 | 2011-03-30 | 彼帕科学公司 | Methods of diagnosing and treating PARP-mediated diseases |
| US20140087970A1 (en) * | 2011-03-30 | 2014-03-27 | Whitehead Institute For Biomedical Research | Serine biosynthesis pathway inhibition for treatment of cancer |
| CN103751221A (en) * | 2014-01-23 | 2014-04-30 | 中国人民解放军第三军医大学第一附属医院 | Application of SIRT1 inhibitor and sorafenib in preparing medicaments for treating liver cancer |
-
2017
- 2017-12-11 CN CN201711307093.2A patent/CN109897866A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101999002A (en) * | 2008-02-04 | 2011-03-30 | 彼帕科学公司 | Methods of diagnosing and treating PARP-mediated diseases |
| US20140087970A1 (en) * | 2011-03-30 | 2014-03-27 | Whitehead Institute For Biomedical Research | Serine biosynthesis pathway inhibition for treatment of cancer |
| CN103751221A (en) * | 2014-01-23 | 2014-04-30 | 中国人民解放军第三军医大学第一附属医院 | Application of SIRT1 inhibitor and sorafenib in preparing medicaments for treating liver cancer |
Non-Patent Citations (1)
| Title |
|---|
| LING TANG等: "Global Metabolic Profiling Identifies a Pivotal Role of Proline and Hydroxyproline Metabolism in Supporting Hypoxic Response in Hepatocellular Carcinoma", 《CLINICAL CANCER RESEARCH》 * |
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Application publication date: 20190618 |
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