CN106636296A - Application of Hsp27 in tolerance diagnosis and treatment for tongue cancer chemotherapy - Google Patents
Application of Hsp27 in tolerance diagnosis and treatment for tongue cancer chemotherapy Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
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- G—PHYSICS
- G01—MEASURING; TESTING
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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Abstract
The invention discloses an application of Hsp27 in tolerance diagnosis and treatment for tongue cancer chemotherapy. The conditions that the Hsp27 protein level in a tongue cancer cell culture supernatant and the Hsp27 protein level in serum of a tongue cancer patient can be raised through chemotherapeutic drug treatment and expression of the Hsp27 in a tolerance cell for tongue cancer chemotherapy is higher than that in a sensitive cell are verified for the first time; and the chemosensitivity of a drug-resistant cell is improved by interfering with and lowering the expression of the Hsp27 and the chemotherapy tolerance of the tongue cancer cell is improved through overexpression of the Hsp27. New data is provided for research on a tolerance mechanism for tongue cancer chemotherapy, a new detection marker is provided for prediction of the tongue cancer chemosensitivity, and a theoretical basis is provided for development of a new treatment strategy based on the Hsp27 as a target.
Description
Technical field
The invention belongs to biotechnology or medical domain, more specifically, the present invention relates to Hsp27 is examined in tongue cancer Chemoresistance
Application in controlling.
Background technology
Heat shock protein is one group of highly conserved protein in evolution, in addition to hyperpyrexia, ischemic, tissue damage, virus
Or bacterium infection etc. also can be produced, it confirms that various critical functions can be played in many fields.In the past people were for heat shock protein
The research of white 27 (Heat shock protein 27, Hsp27) is concentrated mainly on the aspects such as Hsp27 anti-apoptotic, antioxidant radical
Effect (Small heat shock proteins and protection against injury.Ann N Y Acad
Sci.199930；874：66-8.Review)；There is presently no and find that the application to Hsp27 in tongue cancer Chemoresistance diagnosis and treatment has
Understand and in-depth study.
The content of the invention
In view of this, application of the present invention to Hsp27 in tongue cancer Chemoresistance diagnosis and treatment is conducted in-depth research, not only
New data can be provided for tongue cancer Chemoresistance Mechanism Study, and new detection mark is provided for the prediction of tongue cancer chemosensitivity
Will thing, and provide theoretical foundation for the new therapeutic strategy of target spot based on Hsp27 for exploitation.
A kind of applications of Hsp27 in tongue cancer Chemoresistance diagnosis and treatment, in this application, chemotherapy can induce Human Tongue Carcinoma Lines and trouble
Hsp27 is raised in person's serum.
Further, Hsp27 can increase the tolerance of Human Tongue Carcinoma Lines chemotherapy.
Further, extracellular Hsp27 can activate NF- κ B signals in Human Tongue Carcinoma Lines.
Further, extracellular Hsp27 is by being combined activation NF- κ B signals with TLR5.
Further, intracellular Hsp27 can suppress the apoptotic signal that BAX and BIM function inhibitios chemotherapy is induced.
Further, Hsp27 and Tongue Cancer Patients prognosis are in negative correlation.
The beneficial effects of the present invention is：
The present invention confirms that first chemotherapeutics is processed and can raise Hsp27 protein levels and tongue in Human Tongue Carcinoma Lines culture supernatant
Hsp27 protein levels in cancer patients serum, and it is higher than sensitive cells that Hsp27 is expressed in tongue cancer Chemoresistance cell；Under interference
Hsp27 expression is adjusted to increase the chemosensitivity of mdr cell, and overexpression Hsp27 then increases the chemotherapy tolerance of Human Tongue Carcinoma Lines；
In mechanism, extracellular Hsp27 can activate NF- κ B signals and mediate Chemoresistance by TLR5, intracellular Hsp27 suppress BAX and
The apoptotic signal of BIM function inhibitios chemotherapy induction, while finding that the high expression of Hsp27 and Tongue Cancer Patients prognosis are in negative in tongue cancer
It is related.It is so as to the present invention is not only that tongue cancer Chemoresistance Mechanism Study provides new data and pre- for tongue cancer chemosensitivity
Survey and new detection mark is provided, and theoretical foundation is provided for the new therapeutic strategy of target spot based on Hsp27 for exploitation.
Description of the drawings
Figure 1A-Figure 1B is the experimental result picture of embodiment 1；
Fig. 2A-Fig. 2 C are the experimental result picture of embodiment 2；
Fig. 3 A- Fig. 3 B are the experimental result picture of embodiment 3；
Fig. 4 A- Fig. 4 D are the experimental result picture of embodiment 4；
Fig. 5 A- Fig. 5 E are the experimental result picture of embodiment 5；
Fig. 6 A- Fig. 6 C are the experimental result picture of embodiment 6；
Fig. 7 A- Fig. 7 C are the experimental result picture of embodiment 7.
Specific embodiment
Below in conjunction with accompanying drawing, the present invention is clearly and completely described.
Embodiment 1：Chemotherapy can induce Hsp27 in Human Tongue Carcinoma Lines and patients serum to raise
As a result show, Hsp27 protein levels are apparently higher than normal oral mucosa cell in mdr cell Tca8113/PYM
The relatively more sensitive Human Tongue Carcinoma Lines (Figure 1A) of the chemotherapy such as Hacat and Tca8113, SCC-25, SCC-15, SCC-9 and CAL-27, together
When mdr cell nutrient solution in Hsp27 protein levels also apparently higher than Hacat, Tca8113, SCC-25, SCC-15, SCC-9 and
CAL-27 cell culture fluids (Figure 1A)；We process above-mentioned cell, discoveryization with PYM (80mg/L) and cDDP (5mg/L) respectively
Treat in the different degrees of each cell of induction of energy and Hsp27 protein levels raise (Figure 1A) in cell culture fluid；Additionally, we examine
The protein level of Hsp27 in 30 normal healthy peoples and 50 Tongue Cancer Patients serum is surveyed, 50 Tongue Cancer Patients serum include 28
Example head examines patients serum and 22 processes with the patients serum of PYM and/or cDDP chemotherapies, in finding Tongue Cancer Patients serum
Hsp27 protein levels are apparently higher than normal health patients serum, the Hsp27 albumen water in same Tongue Cancer Patients after chemotherapy in serum
The flat Hsp27 expression apparently higher than before chemotherapy (Figure 1B), in the possible rise Human Tongue Carcinoma Lines of prompting chemotherapy itself.
Embodiment 2：Hsp27 increases the experiment in vitro of Human Tongue Carcinoma Lines chemotherapy tolerance
In order to observe effects of the Hsp27 in tongue cancer Chemoresistance, we pass through shRNA technology silences Tca8113/PYM
Hsp27 expression in cell, wherein sh-1# and sh-2# interference effects most substantially (Fig. 2A), still choose sh-1# and sh-2# enters
Row subsequent experimental, set up Hsp27 stable low-expressions clone Tca8113/PYM-sh-1# and Tca8113/PYM-sh-2# and
Control cell lines Tca8113/PYM-sh-con, with MTS experiment detection drug susceptibilities, find to disturb after Hsp27 expression,
Tca8113/PYM cells increase (Fig. 2A) to the sensitiveness of PYM and cDDP；Meanwhile, in order to whether the Hsp27 for observing secretion participates in
The Chemoresistance of Human Tongue Carcinoma Lines, is processed after Tca8113/PYM cells with Hsp27 neutralizing antibodies, can equally increase chemosensitivity
(Fig. 2A).Conversely, the stable overexpression of Hsp27 is set up in Tca8113 and SCC-25 cells by transfection pLEX-Hsp27 carriers
Clone and corresponding control cell lines, after finding overexpression Hsp27, Tca8113 (Fig. 2 B) and SCC-25 (Fig. 2 C) cell pair
The sensitiveness of chemotherapy is reduced；Meanwhile, with restructuring people's Hsp27 albumen (rhHsp27) process also can increase Tca8113 (Fig. 2 B) and
The chemotherapy tolerance of SCC-25 (Fig. 2 C) cell.
Embodiment 3：Hsp27 increases the experiment in vivo of Human Tongue Carcinoma Lines chemotherapy tolerance
Above-mentioned cell in vitro level experiment shows that Hsp27 can participate in the Chemoresistance of Human Tongue Carcinoma Lines, here, we are in nude mice
Experiment in vivo further verifies effects of the Hsp27 in tongue cancer chemotherapy, by Tca8113/pLEX-con and Tca8113/pLEX-
Hsp27 cells are inoculated in the front oxter of nude mice into after knurl, and the 15th day after inoculation starts, and lumbar injection normal saline is given respectively
(0.2ml), PYM (30mg/kg) and cDDP (3mg/kg), per 3 days 1 time, coprocessing 10 times measured the length of a tumour per 3 days
Footpath and wide footpath and calculate gross tumor volume, and be depicted as tumor growth curve, the experiment neck that breaks after terminating puts to death nude mice, peels off swollen
Knurl, measures last volume and weighs.As a result show that overexpression Hsp27 can increase the internal chemotherapy tolerance of Tca8113 cells,
Under chemotherapy regimes, the gross tumor volume of Hsp27 overexpression groups is more than control group (Fig. 3 A), and knurl weight overweights control group (Fig. 3 B).
Embodiment 4：NF- κ B signals in extracellular Hsp27 activation Human Tongue Carcinoma Lines
Detect that GAP-associated protein GAP level finds mdr cell Tca81113/ by the Reporter Gene Experiments and WB of NF- kB activities
NF- kB activities are higher than Hacat, Tca8113, SCC-25, SCC-15, SCC-9 and CAL-27 cell in PYM, meanwhile, Tca8113/
The core p65 protein levels of PYM are higher than NF- κ B classics target gene Survivin and IL-6 in other cells, and Tca8113/PYM
MRNA and protein level are higher than his cell (Fig. 4 A, p of base<0.0001), NF- κ B in mdr cell Tca8113/PYM cells are pointed out
Signal path is activated.Furthermore, it is found that p-I κ B alpha levels are higher than other cells in Tca8113/PYM cells, and total I κ B α albumen water
It is flat then be less than other cells (Fig. 4 A)；Interference silence Hsp27 is expressed and can lower Tca8113/ with the process of Hsp27 neutralizing antibodies
NF- kB activities and core p65 protein levels in PYM cells, while lowering NF- κ B target genes Survivin and IL6 expression (Fig. 4 B, p
<0.0001)；And overexpression Hsp27 and with rhHsp27 process then can raise Tca8113 and SCC-25 cells NF- kB activities and
The nucleoprotein level of p65, and Survivin and IL6 expressions (Fig. 4 C, p<0.0001).Furthermore, in tongue cancer
Hsp27 protein levels are proportionate with p65 core displaced levels, and with I κ B α protein levels in negatively correlated (Fig. 4 C, Fig. 4 D).In order to
The outer Hsp27 of observation of cell whether by activate NF- κ B signals and in Human Tongue Carcinoma Lines function, we are shown using transfection I κ B α
Property negative mutational vector (pBabe-I κ B α) or (suppressed by suppressing I κ B α phosphorylations using NF- kB inhibitor BAY11-7082
NF- κ B signals) process Tca8113 and SCC-25 cells after, then with rhHsp27 processs, find to transfect pBabe-I κ B α and use
BAY11-7082 process can substantially reverse NF- kB activations (Fig. 4 D, p in rhHsp27 induction Human Tongue Carcinoma Lines<0.0001).These
The Hsp27 of experimental result prompting secretion can activate NF- κ B signals in Human Tongue Carcinoma Lines by lowering I κ B α.
Embodiment 5：Extracellular Hsp27 is by being combined activation NF- κ B signals with TLR5
Find that TLR5 protein levels are higher than normal oral mucosa epithelial cell (Fig. 5 A) in Human Tongue Carcinoma Lines after testing；Using
TLR5 expression in Tca8113/PYM cells is lowered in the interference of sh-RNA technologies, and wherein sh-3# and sh-4# interference effects preferably, are chosen
Sh-3# and sh-4# is used for further experiment (Fig. 5 B)；In Tca8113/PYM cells, after interference TLR5 expression, NF- kB activities,
Core p65 protein levels and I κ B α phosphorylation levels are reduced, and total I κ B alpha levels are raised, as NF-KB target genes Survivin and
The mRNA expressions of IL6 reduce (Fig. 5 C, p<0.0001)；In interference Tca8113 and SCC-25 cells after TLR5 expression, then use
RhHsp27 process, it is found that interference silence TLR5 expression can reverse the I κ B α phosphorylations and total I κ B α protein levels of rhHsp27 inductions
Reduce, NF- kB activities and core p65 protein levels are raised and Survivin and IL-6 expression rising (Fig. 5 D, p<0.0001)；
Further, whether we directly can be interacted using co-immunoprecipitation experiment observation Hsp27 with TLR5, use rhHsp27
(10ug/ml) process Tca8113 and SCC-25 cell 1h after, extract total protein of cell, first with Hsp27 antibody protein precipitations after,
Find that Hsp27 can be combined with TLR5 with TLR5 antibody row western blot experiments, similarly, with TLR5 antibody protein precipitations
Afterwards, find that TLR5 can be combined (Fig. 5 E) with Hsp27 with Hsp27 antibody rows westernblot experiments.These experimental result promptings are outer
The Hsp27 of source property can be combined with TLR5, so as to the NF- κ B signals in activation Human Tongue Carcinoma Lines.
Embodiment 6：Intracellular Hsp27 suppresses the apoptotic signal of BAX and BIM function inhibitios chemotherapy induction
In order to observe effects of the Hsp27 in chemotherapy is apoptosis-induced, chemotherapy under the conditions of Hsp27 expression is intervened is have detected first
To the expression of apoptosis correlation molecule and the impact of activity, it is found that no matter whether Hsp27 expression is intervened, even if resistance in Tca8113/PYM
In medicine cell, chemotherapy can induce the expression of BAX, BAD and BIM in Human Tongue Carcinoma Lines, on mitochondria BAX, BAD and BIM level increasing
Plus and cytoplasm in cytochromeC (Cyto C) level rising (Fig. 6 A- Fig. 6 C)；But overexpression Hsp27 substantially suppresses
In Tca8113 and SCC-25 cells on chemotherapy inducing mitochondrial BAX and BIM levels rising, while suppress chemotherapy inducing cell
The rising (Fig. 6 A) of Cyto C levels in matter；In Tca8113/PYM cells, interference Hsp27 expression then increases chemotherapy induction line
The rising of BAX and BIM levels on plastochondria, while increasing the rising (Fig. 6 B) of Cyto C levels in chemotherapy inducing cell matter；In order to
Hsp27 is observed whether by combining BAX and BIM so as to BAX and BIM is shifted to mitochondria under the conditions of suppressing chemotherapy apoptosis-induced,
Find that Hsp27 can be combined with BAX and BIM in Tca8113 and SCC-25 cells by co-immunoprecipitation experiment, and it is combined
Level increases (Fig. 6 C) under chemotherapy regimes as Hsp27 expression increases.These results prompting intracellulars Hsp27 may by with
BAX and BIM is combined and it is shifted to mitochondria under suppression chemotherapy regimes, is induced so as to suppress mitochondria release Cyto invention C
Apoptosis.
Embodiment 7：Hsp27 is with Tongue Cancer Patients prognosis in negative correlation
We have in 84 Jing from attached tumour hospital of Guangzhou medical university (patient has prognosis Follow-up Data)
The protein expression of Hsp27 and its correlation molecule is detected in the tongue cancer of PYM and/or cDDP chemotherapy with ImmunohistochemistryMethods Methods.Send out
Existing 48 tongue cancer example is presented the high expression of Hsp27, and 36 tongue cancers are presented Hsp27 low expressions (Fig. 7 A).SABC shows
Show that TLR5 is generally presented high abundance expression status (Fig. 7 A) in tongue cancer.The no matter height of Hsp27 gene expression abundances, chemotherapy is equal
The expression of BAX and BIM in tongue cancer cell can be induced, and finds that the high expression of Hsp27 is proportionate with the displacement of p65 cores, and and I
κ B α protein levels and Caspase3 activation are in negatively correlated (Fig. 7 A and Fig. 7 B).And prognostic analysis are carried out according to Hsp27 protein levels
It was found that the Tongue Cancer Patients prognosis poor (Fig. 7 C) of the high expression of Hsp27.
The a series of detailed description of those listed above is only for illustrating for the possible embodiments of the present invention,
They simultaneously are not used to limit the scope of the invention, all Equivalent embodiments made without departing from skill spirit of the present invention or change
Should be included within the scope of the present invention.
Claims (6)
1. applications of a kind of Hsp27 in tongue cancer Chemoresistance diagnosis and treatment, it is characterised in that chemotherapy can induce Human Tongue Carcinoma Lines and patient
Hsp27 is raised in serum.
2. applications of the Hsp27 according to claim 1 in tongue cancer Chemoresistance diagnosis and treatment, it is characterised in that Hsp27 can increase
Plus the tolerance of Human Tongue Carcinoma Lines chemotherapy.
3. applications of the Hsp27 according to claim 1 in tongue cancer Chemoresistance diagnosis and treatment, it is characterised in that extracellular
Hsp27 can activate NF- κ B signals in Human Tongue Carcinoma Lines.
4. applications of the Hsp27 according to claim 1 in tongue cancer Chemoresistance diagnosis and treatment, it is characterised in that extracellular
Hsp27 is by being combined activation NF- κ B signals with TLR5.
5. applications of the Hsp27 according to claim 1 in tongue cancer Chemoresistance diagnosis and treatment, it is characterised in that intracellular
Hsp27 can suppress the apoptotic signal that BAX and BIM function inhibitios chemotherapy is induced.
6. applications of the Hsp27 according to claim 1 in tongue cancer Chemoresistance diagnosis and treatment, it is characterised in that Hsp27 and tongue
Cancer patient prognosis is in negative correlation.
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| CN110607298A (en) * | 2018-06-14 | 2019-12-24 | 中国科学技术大学 | 887L RNA inhibitor and application thereof in tumor inhibition |
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| CN110082536A (en) * | 2019-04-17 | 2019-08-02 | 广州医科大学附属肿瘤医院 | A kind of breast cancer cell marker cell factor group and its application |
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Application publication date: 20170510 |