CN102805861A - Lactate dehydrogenase A acetylation activator and application thereof - Google Patents

Abstract

The invention belongs to the field of molecular biology and medicine, and relates to a lactate dehydrogenase A acetylation activator and an application thereof. The lactate dehydrogenase A acetylation activator comprises an LDHA K5 acetylation activator which can be used for effectively activating acetylation of K5. The invention further provides a corresponding antibody and a method for detecting LDHA K5 acetylation. The detection method can be used for detecting the level of LDHA K5 acetylation in a tumor sample, is simple, practicable, rapid and effective, and is low in cost; and the LDHA K5 acetylation is detected by using the method, and inhibition is performed specific to the level of LDHA K5 acetylation, so that a novel simple and convenient way is provided for tumor metabolism diagnosis and treatment.

Description

Lactate dehydrogenase A acetylation activator and uses thereof

Technical field

The invention belongs to molecular biology and medical domain, relate to a kind of lactate dehydrogenase A (LDHA) acetylation activator and uses thereof, also relate to and detect acetylizad antibody of LDHAK5 and detection method.

Background technology

Tumor belongs to metabolic disease and stems from Otto Warburg and propose tumor cell metabolism notion the thirties in eighties of last century; Be that cellular metabolism adjusting (like embryo or tumor) is different from the normal mature cell in the mushroom tissue; Through glycolysis or Warburg effect; Cancerous cell absorbs glucose and produce power and growth (Warburg, 1956) fast than other cells with higher efficient.Research shows; Normal cell only carries out glycolysis under anoxybiotic situation; Even and tumor cell also preferentially carries out glycolysis under not anoxybiotic situation; Consume more glucose and produce more lactic acid, famous Warburg effect that Here it is, therefore Mr. Warburg also obtained Nobel Prize in medicine.Medical circle has been carried out correlational study widely subsequently; More and more evidences shows that cellular metabolism plays important effect in the generation of tumor and development; But the molecular mechanism about it is not clear, and many aspects also are worth deeply inquiring into (Mazurek et al., 2005); More noticeablely be; FDG PET image all shows as increasing and metabolic change of glucose absorption in different types of former and the tumor that shifts; Add the breakthrough of cell signal path research and the innovation of laboratory facilities; Especially the extensive application of protein science research means, the research of tumor metabolic regulation becomes the focus of international research again.

Discover that many metabolic enzymes receive acetylizad regulation and control; Follow-up functional study also shows, the acetylation regulation and control of metabolic enzyme in tumor development, possibly play an important role (Choudhary et al., 2009; Zhao et al., 2010); Lactic acid dehydrogenase catalysis acetone acid under anoxybiotic situation changes into lactic acid, and the while is with the conversion of NADH and NAD+.Research shows that also LDHA plays important effect in the generation of tumor.The rise of the stable LDHA of causing of the HIF-1 α that proto-oncogene such as RAS and Myc cause (Dang and Semenza, 1999; Semenza et al., 2001; Kim JW, 2005), the expression of LDHA is obviously increased in tumor with active; In addition, lactic acid is glucolytic main end-product, and it can produce favourable microenvironment (Koukourakis et al., 2006) and promote tumor-infiltrated (Swietach et al., 2007), and suppresses anticancer immune molecule (Fischer et al., 2007); Interstitial cell can absorb the lactic acid that tumor produces through monocarboxylate transporter body MCT1 and MCT2 and produce acetone acid, thereby keeps tumor cell existence and growth (Koukourakis et al., 2006).Recently Fantin illustrates, and tumor proliferation depends on LDHA (Fantin, 2006) very much, for the effect of LDHA in tumor takes place provides further evidence.Above-mentioned research shows, the important function of LDHA in the tumor cell metabolism.In all tumors, all express LDHA is the focus and the difficult point of puzzled biologist always, and particularly post translational modification and the protein stability thereof about LDHA do not appear in the newspapers in the world.

The inventor's early-stage Study finds that LDHA receives acetylizad regulation and control in the cancer of pancreas tissue sample; The functional study discovery acetylation of carrying out subsequently possibly regulated the active and degraded of metabolic enzyme; Not only in glycolysis, be in the downstream that acetone acid produces based on LDHA; And there is high expressed in nearly all cancer cell in LDHA; And low expression in most of normal cells is again vital to tumor cell proliferation, therefore simultaneously; Researcher thinks that LDHA is the target of potential promising treatment of cancer.

Summary of the invention

The purpose of this invention is to provide a kind of lactate dehydrogenase A acetylation activator.

Further purpose of the present invention provides the purposes of said lactate dehydrogenase A acetylation activator.

Among the present invention, described lactate dehydrogenase A acetylation activator for activating the acetylizad activator of LDHAK5, is characterized in that it contains LDHAK5 acetylation and acetylase and deacetylase described activator.

LDHAK5 of the present invention is arranged in the exons 1 (Homo sapiens1MATLKDQLIYNLIYNLLKEEQTPQNKITVVGVGAVGMACAIS 38) of LDHA, has the sequence like SEQ ID No.1;

Among the present invention, described deacetylase comprises Sirt1, Sirt2, HDAC5, HDAC6, HDAC7,

Described deacetylase causes the deacetylation (as shown in Figure 1) of LDHAK5;

Among the present invention, suppress the acetylation (as shown in Figure 2) that deacetylase activates LDHAK5.

Among the present invention, described acetylase is selected from the sub-PCAF of nuclear receptor coactivity factor 1, and nuclear receptor is assisted activator P300, and histone acetyl based transferase complex GCN5 or carbohydrate-binding protein (=carbohydrate-binding protein, CBP)

Described acetylase can cause the acetylation of LDHAK5; Glucose causes the acetylation (as shown in Figure 3) of LDHA K5;

Among the present invention, detected the LDHAK5 acetylation in the tumor tissues and expressed, testing result shows that the K5 acetylation in the tumor tissues is starkly lower than cancer beside organism.In one embodiment of the invention, the acetylation level of the acetylation site-specific K5 of cancer of pancreas and cancer beside organism shows that the acetylation level of LDHA K5 is starkly lower than cancer beside organism in the cancer of pancreas tissue; In conjunction with result of study for many years, through reducing its acetylation, the autophagy with the molecular chaperones mediation thereby the enzyme that suppresses LDHA is lived, thereby promotion growth of tumor; Wherein, The association study result shows; There is significant difference (p＜0.0001) in the acetylation level of LDHA K5 in 71 routine cancer of pancreas tissues and 38 routine cancer beside organisms; LDHA K5 acetylation level in tumor tissues is starkly lower than cancer beside organism (as shown in Figure 4), and the acetylation level in said site reduces the enzyme that causes LDHA rising alive and expresses and increases, and finally promotes growth of tumour cell; The acetylation level rising inhibitory enzyme in said site is lived and is expressed, and suppresses pancreatic cancer cell growth (as shown in Figure 5).

Result of the test of the present invention has shown that described LDHA can be tumor treatment new target is provided, and the tumor of treatment comprises cancer of pancreas; Simultaneously, can expand tumor is taken place and the understanding of Warburg effect.

The invention provides a kind of lactate dehydrogenase A acetylation activator, the acetylizad activator of especially a kind of LDHA K5, this inhibitor can effectively activate the acetylation of K5;

The present invention further provides a kind of pharmaceutical composition that is used for treatment of cancer, especially to the acetylizad target drug that improves LDHA K5; Described pharmaceutical composition contains LDHA acetylase activator agent safely and effectively and deacetylase or acetylase and pharmaceutically acceptable carrier or excipient; Wherein, described carrier includes, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol and combination thereof;

Described pharmaceutical composition should be complementary with administering mode.

Among the present invention, described pharmaceutical composition can be made into the injection form, as adopting conventional method with normal saline or contain glucose and the aqueous solution of other adjuvant prepares; Described pharmaceutical composition also can adopt conventional method to process tablet and capsule etc.;

Described pharmaceutical composition is made under aseptic condition like injection, solution, tablet and capsule; Wherein, the dosage of active component is the treatment effective dose, like 0.1 microgram/kg body weight-Yue 10 mg/kg body weight every day; In addition, the polypeptide described in the present invention also can use with the other treatment agent.

When using described pharmaceutical composition; Be that the LDHA acetylase of safe and effective amount and deacetylase or its antagonist, agonist are applied to mammal; Wherein safe and effective dosage is usually at least about 0.1 microgram/kg body weight; And in most of the cases be no more than about 10 mg/kg body weight, preferably safe and effective dosage is about 0.1 microgram/kg body weight-Yue 100 mg/kg body weight; Concrete dosage is factor such as considered route of administration, patient health situation also, and skilled practitioners is not exceeding under the situation of its skill and can prepare voluntarily.

Another object of the present invention provides and detects acetylizad antibody of LDHA K5 and the acetylizad detection method of LDHA K5; The acetylizad antibody of described detection LDHA K5 is to detect the acetylation antibody that contains acetylation site-specific K5, and the polypeptide of preparation K5 acetylation antibody is: acetyl-LDHA (K5), (polypeptide: MATLKDQLIYN), have the sequence like SEQ IDNo.2.Also comprise the acetylation antibody (shown in Figure 6) that detects the site-specific antibody of acetylation-anti-K5 in the detection method.

Among the present invention, whether the acetylizad detection method of described LDHA K5 for the acetylation level that detects said site, take place/the recurrence phase with the tumor of the said individuality of auxiliary judgment, may further comprise the steps:

(1) the acetylation antibody of preparation acetylation site-specific K5;

(2) protein of extracting tissue sample/or preparation tumor tissues paraffin section;

(3) detect tumor tissues K5 acetylation level.

In the detection method of the present invention, the polypeptide of preparation K5 acetylation antibody is: acetyl-LDHA (K5), (polypeptide: MATLKDQLIYN), have the sequence like SEQ ID No.2;

In the said detection method, the AC that detects tumor tissues K5 acetylation level is 1: 1000;

In the said detection method, technology such as the protein extraction that relates to, tissue slice, antibody making and SABC all can adopt the routine operation method of this area; Those skilled in the art can carry out protein extraction, tissue slice, antibody making and SABC by described routine operation method.

Detection method of the present invention can be used for the tumor of individuality is detected, and especially cancer of pancreas of individuality etc. is detected.Using the individual tumor of described LDHA K5 acetylation auxiliary monitoring whether to be in generation/recurrence during the phase, also can use the medicament that improves its acetylizad other treatment tumor simultaneously.The acetylizad method of detection tumor LDHA K5 of the present invention can be used for detecting tumor generation or recurrence, and one of the present invention is detected in the instance, and the LDHAK5 acetylation level in tumor tissues is starkly lower than cancer beside organism, and is as shown in Figure 5.

The invention provides a kind of lactate dehydrogenase A acetylation activator, relate in particular to the acetylizad activator of a kind of LDHA K5, this activator can effectively activate the acetylation of K5.The present invention also provides corresponding detecting method, and described detection method can detect the acetylizad level of LDHA K5 in the tumor sample, this detection method is simple, rapidly and efficiently, with low cost; Utilize this method to detect LDHA K5 acetylation level, and suppress, can and treat for tumor metabolism diagnosis simple and direct new way is provided to it.

Description of drawings

Fig. 1 shows that Sirt2 causes the deacetylation of LDHA K5.

Fig. 2 shows that Sirt2 knocks out and causes the acetylation of LDHA K5 to be increased.

Fig. 3 shows that glucose increases the acetylation of LDHA K5

Fig. 4 shows that the K5 acetylation level in the cancer of pancreas is starkly lower than cancer beside organism.

Fig. 5 shows that the K5 acetylation promotes the growth of pancreatic cancer cell.

Fig. 6 is the evaluation figure of K5 acetylation antibody.

The specific embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning; Condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.

Embodiment 1

SABC detects

1) experiment material

Pancreas glandular tissue paraffin section is from Shanghai City Sixth People's Hospital department of general surgery; LDHA acetylation antibody is provided for oneself; LDH buys from company.

2) pattern detection:

Experiment detects 71 routine cancer of pancreas and 38 cancer beside organisms thereof altogether; Every example is collected 3 routine paraffin sections, carries out HE dyeing respectively, and the acetylizad immuning tissue of LDH and LDHAK5 analyzes; And select the different visuals field and painted intensity is analyzed, the T that matches at last check with software.

3) testing result:

As shown in Figure 4, testing result shows that the LDHA K5 acetylation level in the tumor tissues is starkly lower than the other normal structure of cancer.

Experimental result shows that the LDHA K5 acetylation level in the tumor tissues is starkly lower than the other normal structure of cancer, and described LDHA can be tumor treatment new target is provided.The present invention can prepare lactate dehydrogenase A acetylation activator in view of the above, the acetylizad activator of especially a kind of LDHA, and further preparation is used for the pharmaceutical composition of treatment of cancer.Lactate dehydrogenase A acetylation activator of the present invention, the acetylizad activator of especially a kind of lactate dehydrogenase A can effectively activate the acetylation of K5.The present invention also provides corresponding detecting method, and described detection method can detect the acetylizad level of LDHA K5 in the tumor sample, and simple, rapidly and efficiently, with low cost; Utilize this method to detect LDHA K5 acetylation level, and activate to it, diagnosing and treat for the tumor metabolism provides simple and direct new way.

SEQUENCE LISTING

< 110>Fudan University

< 120>lactate dehydrogenase A acetylation activator and uses thereof

<130>

<160> 2

<170> PatentIn version 3.3

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<212> PRT

<213> Homo sapiens

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Met Ala Thr Leu Lys Asp Gln Leu Ile Tyr Asn Leu Ile Tyr Asn Leu

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Leu Lys Glu Glu Gln Thr Pro Gln Asn Lys Ile Thr Val Val Gly Val

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Gly Ala Val Gly Met Ala Cys Ala Ile Ser

35 40

<210> 2

<211> 11

<212> PRT

<213> Artificial

<400> 1

Met Ala Thr Leu Lys Asp Gln Leu Ile Tyr Asn

1 5 10

Claims (10)

1. a lactate dehydrogenase A acetylation activator is characterized in that, contains LDHA K5 acetylation and acetylase thereof and deacetylase.

2. by the described activator of claim 1, it is characterized in that described LDHA K5 is arranged in the exons 1 of LDHA, have sequence like SEQ ID No.1.

3. by the described activator of claim 1, it is characterized in that described deacetylase is selected from Sirt1, Sirt2, HDAC5, HDAC6 or HDAC7.

4. by the described activator of claim 1, it is characterized in that described acetylase is selected from PCAF, P300, GCN5 or CBP.

5.LDHA the acetylizad antibody of K5 is characterized in that, described antibody is the acetylation antibody that contains acetylation site-specific K5, and the polypeptide for preparing this K5 acetylation antibody is: acetyl-LDHA (K5) has the sequence like SEQ ID No.2.

6. one kind is detected the acetylizad method of LDHA K5, it is characterized in that, detects the acetylation level in said site, may further comprise the steps:

(1) the acetylation antibody of preparation acetylation site-specific K5;

(2) protein of extracting tissue sample/or preparation tumor tissues paraffin section;

(3) detect tumor tissues K5 acetylation level.

7. by the described method of claim 6, it is characterized in that the polypeptide of the acetylation antibody of the acetylation site-specific K5 of described preparation is acetyl-LDHA (K5), has the sequence like SEQ ID No.2.

8. by the described method of claim 6, it is characterized in that described tumor tissues K5 acetylation level is that AC is 1: 1000.

9. the lactate dehydrogenase A acetylation activator of claim 1 is treated the purposes in the cancer drug in preparation.

10. by the described purposes of claim 9, it is characterized in that described cancer is a cancer of pancreas.

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