Patent Application
20220073931
The present invention is based on the identification of glutathione S transferase (GSTs) in carcinogenic tumors and a novel treatment for mammals, which inhibits the protein expression of GSTs proteins. The treatment is carried out through the use of antisense oligonucleotides directed to the messenger ribonucleic acids (mRNA) of the GSTM3 and GSTP1, leading to a reduced proliferation of cancer cells and a decrease in tumor progression. In addition, this reduction in proliferation extends to cancers resistant to conventional therapies.
According to the World Health Organization (WHO), cancer is a generic term that designates a broad group of diseases that can affect any part of the body; they are also called malignant tumors or malignancies. A feature that defines cancer is an altered cell division extending beyond its usual limits, being able to invade adjacent parts of the body or spread to other organs, a process called metastasis. Metastases are the leading cause of death from cancer.
The WHO considers that the most common cancers are lung, breast, colorectal, prostate, stomach, liver, esophagus, cervical, thyroid, bladder, non-Hodgkin lymphoma, pancreas, leukemia, kidney, uterine body, oropharynx, cerebral and central nervous system, ovarian, melanoma, gallbladder, larynx, multiple myeloma, nasopharyngeal, laryngopharynx, Hodgkin lymphoma, testicles, salivary glands, vulva, Kaposi sarcoma, penis, mesothelioma, and vaginal.
Therefore, there is a need for drugs and treatments for mammals that have even been diagnosed with cancer.
As an example, one of the most common treatments for cervical cancer includes a chemoradiation therapy based on cisplatin. In addition, this treatment is regularly the only option to treat cervical cancer in advanced stages, and in most cases the disease cannot be eradicated (see Green, J. A., Kirwan, J. M., Tierney, J. F., Symonds, P., Fresco, L., Collingwood, M., & Williams, C. J. (2001). Survival and recurrence after concomitant chemotherapy and radiotherapy for cancer of the uterine cervix: a systematic review and meta-analysis. Lancet, 358(9284), 781-786.
There is a vast literature and patent application documents related to the treatment of cancers, for example, U.S. patent application Ser. No. 15/270,774 of ZHI-MING ZHENG et al., Refers to polynucleotide markers that can be detected two and can be used for the diagnosis of pre-cancers associated with the Human Papillomavirus and cancers associated with the Human Papillomavirus, such as cervical cancer and cervical intraepithelial malignancies as well as methods of treating such cancers. However, said document, like others, does not indicate the treatment of cancers in mammals already diagnosed with them.
Mexican patent application No. MX/a/2014/004285 refers to a method for the diagnosis of cervical cancer comprising performing an electrophoretic polyacrylamide gel run of a serum sample of a subject suspected of having cervical cancer, as well as determining the presence of at least one protein of molecular weight selected from the group of 60 kDa and 50 kDa. This document is intended to diagnose cervical cancer early, but not to treat mammals that have already been diagnosed with cervical cancer.
European Patent No. EP1531843 refers to the hematological study in oncoginecology, which can be used in patients with cervical cancer recurrence to evaluate the effectiveness of anti-tumor treatment and predict the course of the tumor process. The method makes it possible to carry out the selection of the most important rational method of antitumor impact and minimize the development of a number of side effects. Said document does not describe selecting candidates for said treatment to improve its effectiveness.
Patent document No. JP2005189228 provides a method and kit for diagnosing cancers including non-small cell lung cancer, esophageal cancer, laryngeal cancer, pharyngeal cancer, lingual cancer, stomach cancer, kidney cancer, large intestine cancer, cervical cancer, brain tumor, pancreatic cancer and bladder cancer, which are provided with an immunological technique that uses monoclonal antibodies against AKR1B10. Said document claims a protein consisting of the amino acid sequence of SEQ ID NO:1 in a biological sample, or a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO:1 and has an activity of aldocete lactase. The protein is one that has the amino acid sequence shown in SEQ ID NO:1, which is detected by an immunological method. The document refers to diagnosing the types of cancer mentioned there, but not treating a mammal that has already been diagnosed with cancer.
GSTs are a family of enzymes that exhibit various functions, including the detoxification of xenobiotic compounds, evasion of the immune system and inhibition of apoptosis. In various types of cancer, it has been reported that members of the glutathione S-transferase (GST) family are overexpressed and in most cases they are related to a poor prognosis and resistance to chemotherapy (see Cabelguenne et al., 2001; Huang, Tan, Thiyagarajan, & Bay, 2003; Meding et al., 2012; Pectasides, Kamposioras, Papaxoinis, & Pectasides, 2008). In particular, it has been reported that GSTP1 and GSTM3 are deregulated in cancer cells, such as: triple negative breast cancer, prostate cancer, lung cancer and colorectal cancer (see Loktionov, a, Watson, M. a, Gunter, M., Stebbings, W. S., Speakman, C. T., & Bingham, S. a. (2001) Glutathione-S-transferase gene polymorphisms in colorectal cancer patients: interaction between GSTM1 and GSTM3 allele variants as a risk-modulating factor Carcinogenesis, 22(7), 1053-1060.
In addition, it is known that the GSTP1 protein plays a regulatory role through interaction with the TRAF2 protein and decreased signal transduction of the receptors in the TNF-α and JNK pathways, which are responsible for the activation of the apoptosis (see Adler, V., Yin, Z., Fuchs, S. Y., Benezra, M., Rosario, L., Tew, K. D., Ronai, Z. (1999). Regulation of JNK signaling by GSTp. The EMBO journal, 18(5), 1321-1334). On the other hand, it has been observed that the overexpression of GSTM3 in colon cancer is considered a marker of regional lymph node metastases (see Meding, S., Balluff, B., Elsner, M., Schöne, C., Rauser, S., Nitsche, U., . . . Walch, A. (2012). Tissue-based proteomics reveals FXYD3, S100A11 and GSTM3 as novel markers for regional lymph node metastasis in colon cancer. Journal of Pathology, 228(4), 459-470), and the subexpression of GSTM3 in urinary bladder cancer is associated with longer survival (see Mitra, A. P., Pagliarulo, V., Yang, D., Waldman, F. M., Datar, R. H., Skinner, D. G., . . . Cote, R. J. (2009). Generation of a concise gene panel for outcome prediction in urinary bladder cancer. Journal of Clinical Oncology, 27(24), 3929-3937).
Recently antisense molecules capable of inhibiting gene expression with great specificity have been used and, due to this, many research efforts related to the modulation of gene expression by antisense oligonucleotides (OAS) are being made. Some of these OAS focus on the inhibition of specific genes such as oncogenes or viral genes. Antisense oligonucleotides are directed against RNA (sense chain) or against DNA, wherein they form triple structures that inhibit transcription by RNA polymerase II. To achieve a desired effect on the negative regulation of the specific gene, oligonucleotides should promote the decomposition of the targeted mRNA or block the translation of that mRNA, thus avoiding de novo synthesis of the unwanted target protein (see US 20120029060 A1)
International publication No. WO2017091885, which describes the use of antisense oligonucleotides, provides compounds, compositions and methods for modulating the expression of the monocarboxylate transporter 4 (MCT4). In particular, this invention relates to antisense oligonucleotides (OAS) capable of modulating the expression of human MCT4 mRNA and uses and methods thereof for the treatment of various indications, including various cancers. In particular, the invention relates to therapies and treatment methods for cancers such as prostate cancer, including castration-resistant prostate cancer (CRPC).
Protein overexpression of GSTM3 and GSTP1 proteins during tumor progression (hereinafter identified as PT) play a regulatory role through interaction with proteins, for example TRAF2/6 proteins and, therefore, a evasion of signal transduction of apoptosis activation, favoring cell survival and PT (Wu Y, Fan Y, Xue B, Luo L, Shen J, Zhang S, Jiang Y, Yin Z. Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2-ASK1 signals. Oncogene. 2006; 25: 5787-800. Doi: 10.1038/sj.onc.1209576, Although GSTM3 interacts with TRAF6). In addition, GST's expression is involved in the modulation of detoxification processes in cancer cells and, therefore, participates in the chemoresistance response of conventional therapies in the cancer patients.
The present invention relates to the identification of GSTM3 and/or GSTP1 proteins in cancers, to provide a treatment with antisense oligonucleotides directed to said proteins (GSMT3 and GSTP1) to mammals identified as candidates for said treatment. At least one or more of said combined oligonucleotides block a protein specifically or both proteins.
According to a first aspect, the invention is directed to GSTM3 and GSTP1 proteins to be used as therapeutic targets and/or prognostic factors for mammals with cancer.
In the present invention, xenografted cancer cell lines in immunosuppressed mice were used to analyze, through their proteomics, the differences in protein expression during tumor progression. In this analysis it was found that glutathione S transferase P1 and M3 (hereafter referred to as GSTP1 and GSTM3, respectively) were some of the proteins that consistently increased their levels during tumor growth. In addition, it was found that through “inhibition of genes” (knock-down) of said proteins, they play a critical role for cell survival and tumor progression. Also, the abundance of the levels of these proteins in cancer biopsies was correlated with the survival of the patients. Therefore, it was shown that GSTP1 and GSTM3 proteins are also useful for prognostic purposes and that they are excellent candidates for target gene therapy for cancer.
In a second aspect, the present invention provides the use of antisense oligonucleotides of glutathione S transferases, preferably GSTM3 and GSTP1, without being limited thereto, for the treatment of cancers, in candidate subjects who have previously been diagnosed with cancer, wherein antisense oligonucleotides are between 15-50 nucleotides in length.
In another aspect, the present invention provides a method of treatment for cancer comprising: a) the extraction of the protein from the tumor tissue, b) carrying out an analysis by immunodetection techniques such as, for example, spotting of bands by western (Western blot), immunohistochemistry, ELISA, etc., to identify if the tumor has GSTM3 and/or GSTP1 proteins and c) administer the antisense oligonucleotides for said proteins.
In another aspect, the present invention provides a kit for identifying a candidate subject to be treated with the oligonucleotides of the present invention comprising at least one antisense oligonucleotide of the glutathione S transferases, preferably GSTM3 and GSTP1, without being limited thereto., a protein extraction solution, at least two antibodies for the identification of GSTM3 and GSTP1 proteins and optionally a secondary antibody, and a colorimetric developing solution for western (western blot) or immunohistochemistry (IHQ) staining.
Additional aspects and advantages of the present invention will be better understood by persons skilled in the art in light of the detailed description and with reference to the following figures.
As indicated above, the word cancer is a generic term that designates a broad group of diseases that can affect any part of the body, such as lung cancer, breast cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, esophageal cancer, cervical cancer, thyroid cancer, bladder cancer, non-Hodgkin lymphoma, pancreatic cancer, leukemia, kidney cancer, uterine body cancer, oropharyngeal cancer, brain and central nervous system cancer, ovarian cancer, melanoma cancer, gallbladder cancer, larynx cancer, multiple myeloma cancer, nasopharyngeal cancer, laryngopharyngeal cancer, Hodgkin lymphoma, testicular cancer, salivary gland cancer, vulvar cancer, Kaposi sarcoma, penile cancer, mesothelioma, and vaginal cancer, among others, so it should be understood that a person skilled in the art will appreciate that the invention described below is susceptible to variations and modifications different from those specifically described and therefore, the present invention includes all such variations and modifications as well as all the stages, features, compositions and compounds referred to or indicated therein, whether in individual or collective and any of all combinations or any of two or more of the stages or features.
Furthermore, it should also be understood that the present invention is not limited in scope to the specific embodiments described therein, which are proposed for exemplification purposes only. Functionally equivalent products, compositions, combinations and methods as well as their application in mammals are clearly within the scope of the invention, as described.
Tumor progression (hereinafter also referred to as PT) involves changes in the deregulation of metabolic and cellular processes. The study of tumor proteome dynamics represents the protein changes of this deregulation mechanism during PT. Therefore, the study of proteome dynamics for example in cervical cancer (hereinafter also referred to as CC) will provide relevant information to understand PT and the disease to be treated.
Protein overexpression of the glutathione S transferase GSTM3 and GSTP1 genotypes during tumor progression (PT) plays a regulatory role through interaction with proteins, for example TRAF2/6 and therefore an evasion of transduction of signals of apoptosis activation, favoring cell survival and PT. In addition, GST expression is involved in the modulation of detoxification processes in cancer cells and, therefore, participates in the survival response to conventional chemotherapy in patients with CC and other types of cancer.
The present invention relates to antisense oligonucleotides of glutathione S transferase (GSTs) such as GSTM3 and GSTP1, as novel candidates for use as therapeutic targets and/or prognostic factors for patients (mammals) who have been diagnosed with CC and other types of cancer.
The identification of candidate subjects for the treatment of cancers and their treatment is carried out by identifying the protein expression of glutathione S transferase proteins (GSTs). Treatment includes the use of antisense oligonucleotides targeting the messenger ribonucleic acids (mRNA) of the GSTM3 and GSTP1, which give tumor cells greater resistance to chemotherapies.
The antisense oligonucleotide (OAS) is preferably any antisense oligonucleotide, which reduces the expression levels of the GSTMs and thus increases the sensitivity of the cell, tissue and/or organ to the chemotherapeutic agent in vitro, ex vivo, or in vivo.
In a first embodiment, the antisense oligonucleotide is an oligonucleotide composed of subunits called “nucleotides”, wherein each nucleotide is made up of three parts: a sugar or a functional analogue thereof, a nitrogen base and a functional group that serves as an internucleotide bond (usually a phosphate group) between the subunits that make up the oligonucleotide. Each of these components may contain the following modifications:
wherein
X means a sugar, ribose in the case of RNA or deoxyribose in the case of DNA or the functional analog thereof in the nucleotide.
B means the nitrogenous base bound to sugar or a functional analogue thereof, and R means the functional group in carbon 2′ of sugar when it is ribose.
In a second embodiment, modifications to ribose sugar, without being limiting, are:
Modifications in the 2′ position of ribose sugar that include the replacement of the OH group by different groups among which are (2′-O-methyl (2′OMe), 2′-O-methoxyethyl (2′-OMOE), 2′fluor (2′-F) and 2′-chiral fluor (2′-F))
wherein base refers to the nitrogenous base of the nucleotide.
In a third embodiment, modifications in ribose include the use of an analogue of bicyclic ribose, wherein the ribose structure contains an additional ring, for example, ribose with 2′-O, 4′-C-methylene (LNA-blocked nucleic acid), 2′-O, 4′-C-oxymethylene, 2′-O, 4′-C-methylene-β-D-ribofuranosil rings, among others.
Additionally, the substitution of ribose sugar can be carried out by analogous functional groups with similar function, for example, the substitution of the ribose by another sugar such as arabinose, morpholino, or by ribose analogs with a spirocyclic ring in different positions of the sugar ring, without being such limiting examples.
The following are examples of some of the different modifications to the sugary part of the oligonucleotide.
In a fourth embodiment, modifications to the nucleobases or nitrogenous bases include, but are not limited to, adenine, cytosine, guanine, thymine, as well as their modifications 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2-diaminopurine, hypoxatin, 5-propynyl uracil, 2-thio thymine, N3-thioethyl thymine, 3-deaza adenine, 8-azido adenine and 7-deaza guanine, or the use of universal bases such as 3-nitropyrrole, imidazole-4-carboxamide, 5-nitroindole.
The following are examples of some structures of different types of modified nucleobases and nucleobase analogs.
In a fifth embodiment, the modifications in the column or skeleton of the internucleoside groups, which bind them, can be but are not limited to:
5′-N-carbamate, methylene-methylimine, amide, phosphorodithioate, thioether, thioformacetal and mercaptoacetamide. The substitution of the phosphodiester or phosphorothioate group can be carried out by phosphorodiamidate groups with piperazine and morpholino residues (PMOs).
For a person skilled in the art of the present invention it will be obvious that, within the scope, the chimeras resulting from the mixture of 2 or more chemical modifications mentioned above are also included, for example without being limiting, the replacement of the ribose by a morpholino ring and replacement of the phosphodiester group with phosphorodiamidate groups without charge (PMOs), the replacement of ribose and the phosphodiester bond is replaced by pseudopeptide N-(2 aminoethyl) glycine and the nucleobase is linked to the oligonucleotide column through of an ethyl enecarbonyl bond (PNA), a chimera where the ribose contains an alkyl substituent at the 2′ position and the phosphodiester bond is replaced by a phosphotiester bond, a chimera wherein the ribose is replaced by a ribose analogue with 2′-O,4′-C-methylene (LNA) rings and the phosphodiester bond is substituted by a phosphotiester bond, among others.
Examples of chimeras resulting from the combination of the different components of a nucleotide are by way of example:
In order to create a suitable model to study tumor progression (PT), cervical cancer cell lines (SiHa and HeLa), triple negative breast (MDA-231-MB) and colon (COLO 205) were used to generate grafted tumors in mice. Cancer cells were cultured at 70% confluence and 107 cells were inoculated into female mice of the Nu/Nu strain for 4 to 6 weeks. The tumor volume was measured according to the equation of the volume of an ellipsoid described as: Vtm=η/6 (L*W*H), where L: length, W: width and H: height, and were taken in seven different moments of PT, for example, a kinetics of HeLa and SiHa tumors was taken at 5, 10, 15, 20, 30, 45 and 50 days after inoculation. The first four measurement times showed a low progression of tumor growth. However, at the endpoints of kinetics, tumor volume grew exponentially for HeLa cell tumors. From day 30 to 45 the average tumor volume doubled and by day 50 the average volume was 3 times more than the previous measurement. For SiHa cells, tumor growth rates were lower than HeLa; from 30 to 45 days, SiHa tumors grew 1.6 times more, while, in the last five days, tumors grew on average 1.6 times larger (see
Analysis of 2D-PAGE Gels and Protein Identification.
Tumors of the two types of cancer cells (HeLa and SiHa) were collected and total proteins were extracted for analysis by means of two-dimensional electrophoresis gels (2D-PAGE). The analysis of the 2D-PAGE images of each repetition for each time studied and for each type of cell was carried out using the PDQuest software. An average of 866 electrophoretic entities (spots or “stains”) were detected for samples of HeLa tumors at each repetition. For SiHa tumors, the average of spots or “stains” detected in 2D-PAGE images was 766. The correlation coefficient between repetitions for each tumor time and cell type was determined from the 2D-PAGE maps.
Table 1 below shows that the correlation coefficient in all tumors was greater than 0.7 in both types of cells. The proteomic profile was obtained each time and then compared to find differentiated proteins during PT.
In addition, 601 points were detected in HeLa tumors in the three times of PT, while in SiHa tumors the number of common points was 716 (see
Table 2 below shows that 46 different proteins were identified from HeLa tumors, including 34 with constant expression through PT, 7 proteins showed a negative regulation along PT, 3 proteins increased their abundance during tumor growth, and 2 were found with an oscillating pattern.
Table 3 below shows that a total of 44 proteins were identified from the SiHa cells. The identified proteins were distributed according to their expression pattern, 20 were found without differences in the three tumor ages evaluated, 8 decreased their abundance during PT, while 16 showed an increasing pattern. When analyzing all the proteins identified in tumors of both types of cells (Hela and SiHa), it was found that 34 proteins were shared between the two types of tumors, including 14 that show the same pattern of expression.
Among the proteins with levels of overexpression, two family members were identified Glutathione S-transferase (GSTM3 and GSTP1). GSTM3 was identified in HeLa tumors and GSTP1 in SiHa tumors (see
Bioinformatic Analysis
Subsequently, the proteins identified in both tumors were used to perform a functional enrichment analysis based on the biological processes of gene ontologies (Gene Ontology: GO.
The proteins were grouped according to their expression levels and subjected to an enrichment analysis. The results indicated that proteins that increase their levels during PT are mainly involved in anti-apoptotic, cell division, glycolysis, angiogenesis, viral reproduction and regulation of apoptotic processes (see Checa-Rojas A., et al. GSTM3 and GSTP1: novel players driving tumor progression in cervical cancer, Oncotarget. 2018; 9: 21696-21714). In addition, including all identified proteins, the results suggest that during PT the overrepresented pathways are related to the cellular response to stress, the MAPK6/MAPK4 and NIK/NF-kappaB signaling pathways.
On the other hand, data mining analysis revealed that GSTM3 and GSTP1 interact with the proteins of factors associated with the tumor necrosis factor receptor (TRAF). However, said analysis can be extended to other proteins. Specifically, the interaction of GSTP1 with TRAF2 previously validated in HeLa cells (Wu, Y., Fan, Y., Xue, B., Luo, L., Shen, J., Zhang, S., . . . Yin, 2. (2006). Human glutathione S-transferase P1-1 interacts with TRAF2 and regulates TRAF2—ASK1 signals. Oncogene, 25, 5787-5800), and GSTM3 was reported as an interactor of TRAF6 (tumor necrosis factor receptor associated with factor 6) (Rouillard, A D, Gundersen, G W, Fernandez, N F, Wang, Z., Monteiro, C D, McDermott, M G, & Ma'ayan, A. (2016). The harmonizome: a collection of processed datasets gathered to serve and mine knowledge about genes and proteins. Database, 2016, baw100.) (see
To demonstrate that this interaction occurs under physiological conditions, TRAF6 expression was observed in both CC tumors (see
Modulation of MAPK Signaling During PT
Given the importance of TRAF proteins on the downstream activation of the mitogen-activated protein kinase (MAPK) cascade, the phosphorylated version of NF-κB p65 (ser529), ERK, JNK and p38 by Western spotted (blot) of the PT proteins. The results showed that the phosphorylation of p38 and JNK was reduced over time in both CC tumors, but not in pNF-κB and pERK (see
Endogenous Secreted TLR4 Receptor Activators in the CC
On the other hand, it is known that the activation of the TLR4 pathway is driven by the presence of lipopolysaccharides (LPS) from bacterial infections, but also by endogenous activators such as HSP60 and HSP70 thermal shock proteins. To demonstrate that CC cell lines can express endogenous activators of the Toll type receptor 4 (TLR4), an in vitro analysis of the secreted proteins was performed using the HeLa and SiHa cell lines (see
Secreted proteins were analyzed by LC-MS/MS and a total of 432 HeLa and 447 SiHa proteins were identified, of which 264 were common between both cell lines (see
Proteomic mass spectrometry data has been deposited in ProteomeXchange Consortium via the deposit of its associated PRoteomics IDEntifications (PRIDE) with the data set identifier PXD005466.
STM3 Interacts with HPV18 E7
Because in Mileo, A. M., Abbruzzese, C., Mattarocci, S., Bellacchio, E., Pisano, P., Federico, A., . . . Paggi, M. G. (2009). Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival. PLoS ONE, 4 (10) is shown that GSTP1 protein interacts with HPV16 E7 protein and this interaction improves cell survival, to know if GSTM3 can interact with HPV 18 E7, an alignment was performed of structural overlap between the GSTP1 and GSTM3 proteins and the E7 proteins of HPV 16 and 18 using the MAMMOTH program, followed by the Swiss PDB Viewer (Deep View) v4.1 software to visualize the results (see
To demonstrate this interaction, a vector construct was generated to express a GSTM3 recombinant human protein with a histidine tag added at the N-terminal (N-6× His-tag) in S. cerevisiae (see
CACCATCACCACCATCAT TCGTGCGAGTCGTCTATGG
GSTM3-HindIII. This oligonucleotide contains the HindIII restriction site (in bold), yeast consensus sequence at the translational start site and codons for 6 histidines (bold and underlined).
GSTM3-BamHI. This oligonucleotide contains the BamHI restriction site (bold).
Primers used for amplification and cloning of the HPV18 E7 gene
GAT GGT GAT GAT G CT GCT GG
E7-18-Hind III. This oligo contains the Hind III restriction site (bold) and initial HPV 18 sequence (bold).
E7HPV18-his. This oligo contains a fragment of 6× histidine sequence (bold).
Univ His-Tag BamHI. This oligo contains the BamHI restriction site (bold) and 6× histidine sequence fragment (bold and underlined).
Once the interaction of the GSTM3 protein with the HPV18 E7 protein was demonstrated, the relevance of this interaction in cell survival was evaluated. For this purpose, a stress sensitivity test with UV was developed in a breast cancer cell line MDA-MB-231 which is negative for HPV, and the expression of GSTM3 and GSTP1 proteins (see
“Inhibition of Genes” (Knock-Down) GST In Vitro and In Vivo Using Antisense Oligonucleotides
As an example, without being limiting, the antisense oligonucleotides of vivo-morpholinos were designed to inhibit the therapeutic targets of the GSTs starting from the 5′UTR region of the messenger RNAs of the GSTM3 and GSTP1 and the ATG start codon and include 25 nucleotides, but this region is not limiting being able to use a range of 15-50 nucleotides, preferably from 18 to 30, more preferably from to 25, and bases 1-773, preferably close to the start codon, of the GSTP1 gene and for GSTM3 from base 1-4144, preferably close to the start codon, with a similarity of 100-50% of both sequences.
It is obvious that a skilled person in the art can employ any chemical modification of the RNA or DNA sequences to inhibit GSTs, for example: 2′MOE, 2′MO, PNA, LNA, Phosphorothioate, 2′-F, etc. They are commercially available. The preferred GSTs in the present invention, without being limiting thereof, are GSTM3 and GSTP1 with the sequence of antisense oligonucleotide (OAS) 5′-TAGACGACTCGCACGACATGGTGAC-3′ (56% CG−) and 5′-AATAGACCACGGTGTAGGGCG-3G′ (56% CG), respectively.
For both in vitro and in vivo tests both antisense oligonucleotides were dissolved in sterile PBS saline phosphate buffer at pH 7.5.
In order to evaluate the effect of GSTM3 and GSTP1 on CC cell lines, the expression of both proteins was inhibited by means of antisense oligonucleotides (OAS) and a random sequence was used as a control. To evaluate the inhibition of GSTs, eight doses were evaluated for each antisense oligonucleotide (OAS) in culture with two cell lines, HeLa and HaCaT (negative control). The concentrations used were between the range of 10 to 1,280 ng/mL and were incorporated into the culture medium. Subsequently, cell proliferation was evaluated at three different times at 24, 48 and 72 hours. It was observed that HaCaT cells were not affected by treatment with any antisense oligonucleotide during the analysis period. However, a slight loss of survival was noted with the highest dose (1,280 ng/mL). In HeLa cells, viability losses were observed after 48 hours of treatment in all doses of OAS-GSTM3 (see
To evaluate the cellular response in other cell lines, the dose of 640 ng/mL was selected, because this is the highest dose that did not affect the HaCaT cell line. In addition, treatment was performed with 640 ng/mL in the SiHa CC cell line (see
For in vivo assays, 15 days after tumor inoculation in mice, six doses of 400 ng/500 μL were injected intratumorally, every third day. On day 30, tumors were collected for later analysis. A randomized antisense oligonucleotide was used as a control in both in vitro and in vivo assays. The sequences of the antisense oligonucleotides used in
Loss of GST Inhibits Tumor Progression in Cervical Cancer
To demonstrate the importance of GST during tumor progression (PT), the effects of treatments with antisense oligonucleotides in a murine model were examined (see
In CaLo tumors, both proteins expressed as GSTM3 and GSTP1 were found (see
In addition, the response to the treatment of tumors of two cell lines of different origins, MDA-MB-231 of breast cancer and COLO of colon cancer, was also studied. Both tumors exhibited low GSTP1 expression compared to CC tumors (see
GSTM3 and GSTP1 Regulate the MAP Kinase Proteins pJNK and pp38.
The effects of the deactivation of GSTM3 and GSTP1 on the activation of pJNK and pp38 and the phosphorylation of p65 and pERK (from the NF-κB pathway) during CC PT were analyzed (see
GSTM3 and GSTP1 Regulate Cell Survival by Inactivating NF-κB and pERK
The inactivation of the ERK protein and p65 NF-κB was examined (see
In CaLo tumors, only pERK was inactivated after treatment with any of the antisense oligonucleotides for GST (see
GST Expression Analysis in Tissue Samples from CC Patients
A follow-up study of 13 patients with CC who had undergone chemotherapy was performed (see
The results obtained indicate that patients with cervical cancer expressed the GSTM3 and GSTP1 proteins, and that the expression of the tissue samples is related to survival. Therefore, it is concluded that the increase of these proteins is involved with the prognosis of the patient being unfavorable is overexpression.
The present invention provides evidence for the identification and inhibition of the expression of GSTM3 and GSTP1. In the results of the cultures, the cervical cancer cells were drastically affected by the blockade of both GST, while the HaCaT (non-cancerous) control cells were not affected by the inhibition of these proteins, whereby the GSTM3 and GSTP1 are crucial for the survival and proliferation of cancer cells in culture. In inoculated tumors, it was observed that in those CC cell lines expressing at least one of these proteins, the tumor volume decreased dramatically after treatment with antisense oligonucleotides.
The present invention demonstrates that inhibition of GSTM3 or GSTP1 activates the signaling of JNK and p38, which leads the cells to apoptosis and, therefore, decreases the tumor volume. On the other hand, it was observed that the inactivation of NF-κB and/or ERK after GST inhibition inhibits cell survival.
The present invention shows that there is a strong association of the overexpression of GSTM3 and GSTP1 proteins and the survival of patients. The results obtained in vitro and in vivo are consistent with the clinical data, since the survival of patients with CC was associated with high levels of GST protein (see
The following examples will allow us to understand the present invention even more, as well as to show the best method of carrying it out. It should be understood that said examples are illustrative of the present invention and not in any way limiting the scope thereof. References cited herein should be construed as being expressly incorporated therein.
It should also be understood that the methods and techniques of protein extraction, and immunodetection, as well as the protocols for sample preparation, preparative two-dimensional gel electrophoresis, image analysis and protein identification through MALDI mass spectrometry, which are not specifically described in the examples, are reported in the aforementioned literature and are known to those skilled in the art. For example, phenolic protein extraction is described in Hurkman, W. J., & Tanaka, C. K. (1986).
Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant physiology, 81(3), 802-806; Encarnación, S., Guzmán, Y., Dunn, M. F., Hernández, M., Vargas, M. del C., & Mora, J. (2003). Proteome analysis of aerobic and fermentative metabolism in Rhizobium etli CE3. In Proteomics (Vol 3, bll 1077-1085), Klose, J., & Kobalz, U. (1995). Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis, 16(6), 1034-1059.
Tumor Generation
Female athymic nude mice 4-6 weeks old (BALB/c Nu/Nu) were used and subcutaneously injected with 10′ tumor cells in 500 μL of RPMI 1640 medium without FBS and collected in 30, 45 and 50 days. The tumors were measured using a Vernier caliper, and the tumor volume was obtained by calculating the volume of an ellipsoid as π/6 (L*W*H), wherein L: length, W: width and H.
Tumor Protein Extraction, Proteomic Analysis and Mass Spectrometry.
Tumor tissue samples were extracted by macerating them in liquid nitrogen and a cocktail of protease inhibitor and phosphatases, followed by sonication on ice, to then carry out the extraction of phenolic proteins by extraction with RIPA buffer. Subsequently, the protein expression analysis is performed using anti-GSTM3 and anti-GSTP1 antibodies and then visualized by immunodetection techniques such as: western staining (western blot), immunohistochemistry, ELISA, etc.
Extracellular Protein Extraction In Vitro and Ex Vivo.
Cell lines were cultured with RPMI 1640 advanced serum free up to 70-80% confluence. The medium was removed and rinsed three times with sterile saline: 0.9% NaCl (w/v). After washing, FBS-free RPMI 1640 medium without fresh red phenol (Gibco) was added and incubated for 20 h. Later, the medium was recovered and centrifuged at 2,000 g for 5 minutes. The supernatant was passed through a 0.22 μm pore size PVDF membrane (Millex, Millipore) and stored at −70° C. until later use. For extracellular proteins from xenograft tumors, HeLa and SiHa tumors were inoculated with 107 cells. After 30, 45 and 50 days after inoculation, the tumors were collected and washed 3 times with saline. The procedure followed to extract secreted proteins from tumors was performed as previously described for the cells in culture and the supernatant was stored at −70° C. until later use.
Identification of Secreted Proteins Through LC-MS/NS
The identification of secreted proteins in cell lines was separated in SDS-PAGE. The generated peptides were analyzed in a nanoLC-MS/MS system (Q-TOF Synapt G2 MS; Waters), the identification of the peptides and proteins was done through the MASCOT Distiller interface (Matrix Science), and the database Swiss-Prot and NCBI.
Signal Peptide Analysis
For the analysis of the signal peptide a bioinformatic program called SignalP 4.1 was used, which predicts the presence and location of signal peptide sites in amino acid sequences. The method predicts and identifies the export sites of the signal peptide based on physicochemical features and a combination of neural networks (NN) and hidden Markov models (HMM).
Coimmunoprecipitation and Immunostaining (Immunoblot)
The HeLa tumor was collected at 50 days and stored at 80° C. until use. After the tumor sample, they were macerated in liquid nitrogen and lysed with 500 μL RIPA buffer (10 mM Tris, 1 mM EDTA, 1% NP40, 0.1% sodium deoxycholate, 140 mM NaCl) and supplemented with inhibitors of protease and phosphatase (10 mM β-glycerophosphate, 10 mM Na3VO4, 10 mM sodium fluoride). Total cell lysates were centrifuged at 13,000 g for 5 min to settle the insoluble material. The lysates are incubated 2 hours with sepharose protein A, normalized for the total protein concentration (10 μg protein) using SDS-PAGE. Protein candidate antibodies (GSTM3 and TRAF6) were immunoprecipitated by incubating lysates with 6 μL antibody conjugated sepharose overnight at 4° C. The beads were washed 3 times with 500 μL lysis buffer. Co-immunoprecipitant proteins were resolved in 12% SDS-PAGE. GSTM3 and TRAF6 levels were detected by immunoblotting using previously described anti-antibodies.
Commercial antibodies to analyze Western staining (western blot) proteins were selected from anti-GSTM3 (Abcam, ab67530, 1:10,000), anti-GSTP1 (Abcam, ab53943, 1:10,000), anti-TLR4 (Biolegen, 312804, 1:10,000), anti-TRAF6 (Abcam, ab13853, 1:10,000), anti-NF-Kb P65 (SC-378, 1:1,000), anti IKB-α (SC-371, 1:1,000), anti-JNK (sc-1648, 1:1,000), antiERK (sc-94, 1:1,000), anti p38 (sc-535, 1:1,000), anti-NF-κB phospho p65 (sc-101752, 1:1,000), anti-phospho-JNK (sc-6254, 1:1,000), anti-phospho-ERK (sc-7383, 1:1,000), phospho-p38 (sc-7973, 1:1,000), anti-phospho-IKB-α (Cell signaling, 1:1,000), anti-HSP70 and HSP60 (Biolegen, 648005 and 681502, 1:10,000), HPV18 E7 (Abcam, ab38743, 1:1,000), anti-His tag antibody (Invitrogen, 372900, 1:5,000). The cells are lysed in a buffer containing 100 mM Tris (pH 8.6); 4% SDS; 100 mM DTT; a protease inhibitor cocktail. To ensure lysis, pulses are given with a sonicator for 1 second for DNA fragmentation. The proteins are electrophoresed in SDS-PAGE from 12% to 15% and transferred to nitrocellulose membranes using a semi-dry system. The already transferred membranes are blocked with 5% skim milk or bovine serum albumin in a Tris saline buffer containing Tween 20 (TBST) for 15 minutes at 4° C., washed three times in TBST. Subsequently, albumin or skim milk is removed and washed 3 times with TBST, then the membrane is incubated with the primary antibody (anti-GSTM3 or anti-GSTP1) at 4° C. overnight and tested with primary antibody diluted and incubated at 4° C. overnight. After removing the primary antibody the membranes were incubated with a secondary antibody conjugated to peroxidase for 2 h and then the membrane was revealed with a solution of Carbazol (27.2% Carbazol Stock, 72.6% acetate buffer, 0.2% H2O2), Carbazol Stock: N,N-Dimethylformamide≥98% and 3-Amino-9-ethylcarbazole (Sigma-Aldrich) in a 1:8 (w/v) ratio to generate a red/brown. Relative quantifications were performed with ImageJ software.
To analyze tissue samples by immunohistochemistry (IHC), the percentage of the region of interest (ROI) that is the region that gives positive expression of GSTs is analyzed. The medical records were reviewed, taking into account the patient's previous medical history. All cases were subjected to an immunohistochemical analysis using anti-GSTM3 (Abcam, ab67530, 1:1,000) and, anti-GSTP1 (Abcam, ab53943, 1:1,000). The paraffin blocks were sampled at a tissue thickness of 5 μm and produced in duplicate for each slide. The analysis was performed in an automated immunocontainer (Ventana Medical Systems). Three parts of the tumor were evaluated separately in each sample, as was the presence of staining in the tumor cells. In this study, we analyzed the percentage of the region of interest (ROI) stained by the antibodies, as estimated using the CellSens (Olympus) software. The samples were divided into two groups to assess the association of protein expression and patient survival: (W-M) consisting of a weak ROI for GSTM3 and moderate ROI for GSTP1; and (MH-H) which groups a moderate/high ROI for GSTM3 and high ROI for GSTP1. Kaplan-Meier survival curves were used for this analysis using XLSTAT, with the Greenwood IC and a significance level of 95%.
In order to assess the effect of GSTM3 and GSTP1 on CC cells, the expression of both proteins was inhibited by the use of antisense oligonucleotides (OAS). Three OAS were designed, two to specifically block proteins and one with a random sequence as a negative control (OAS-GSTM3, OAS-GSTP1 and OAS-Control). First, eight doses were evaluated for each OAS in culture with two cell lines, HeLa (cervical cancer) and HaCaT (negative cancer control). The doses used were from 10 to 1,280 ng/mL total incorporated into the culture medium. And subsequently, we evaluate cell proliferation at three different times at 24, 48 and 72 hours. In this experiment, it was observed that HaCaT cells were not affected by treatment with OAS-GSTM3 during the analysis period. Only a slight loss of survival was noted with the highest dose (1,280 ng/mL). In HeLa cells, viability losses were noted after 48 hours of treatment in all doses of OAS-GSTM3 (see
Once the protein expression of the GSTM3 and/or GSTP1 in the tumor tissue is identified, the specific antisense oligonucleotides are administered (for example: 2′O-Me, 2′O-MOE, vivo-Morpholino, Morpholinos, LNA, PNA, among others) to perform the silencing of GSTs proteins, which will induce tumor cell death.
Although the present invention has been described with particularity in accordance with certain of the preferred embodiments, the examples should be interpreted only as illustrative of the invention and not for the purpose of limiting it. References cited herein are expressly incorporated for reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| MX/A/2019/001389 | Jan 2019 | MX | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/MX2020/000003 | 1/30/2020 | WO | 00 |
