Analysis of transcription profile to reveal altered signaling pathways following the overexpression of human desumoylating isopeptidase 2 in pancreatic cancer cells.

Human desumoylating isopeptidase 2 (DESI-2) is a member of the DESI family and contains a conserved PPPDE1 domain. Previous studies have demonstrated that DESI-2 overexpression may induce cell apoptosis. In the present study, differentially expressed genes were analyzed using a transcription microarray in DESI-2 overexpressing PANC-1 pancreatic cancer cells. A total of 45,033 genes were examined by microarray, which identified 1,766 upregulated and 1,643 downregulated genes. A series of altered signaling pathways were analyzed, in which certain essential signaling factors, including retinoid X receptor (RXR), BH3 interacting-domain death agonist, Ras homolog gene family member A (RhoA) and Rho-associated protein kinase, were further investigated at the protein level. The release of cytochrome c and the activation of caspase-3 were also detected by western blot analysis. Immunohistochemistry further revealed the expression features of RXR and RhoA in pancreatic ductal adenocarcinoma tissues with various DESI-2 expression levels. The results serve as a valuable reference for the further elucidation of the functions of DESI-2 in pancreatic cancer.

Fully human HER2/cluster of differentiation 3 bispecific antibody triggers potent and specific cytotoxicity of T lymphocytes against breast cancer.

The use of a bispecific antibody (BsAb) is a promising and highly specific approach to cancer therapy. In the present study, a fully human recombinant single chain variable fragment BsAb against human epidermal growth factor receptor (HER)2 and cluster of differentiation (CD)3 was constructed with the aim of developing an effective treatment for breast cancer. HER2/CD3 BsAb was expressed in Chinese hamster ovary cells and purified via nickel column chromatography. Flow cytometry revealed that the HER2/CD3 BsAb was able to specifically bind to HER2 and CD3­positive cells. HER2/CD3 BsAb was able to stimulate T-cell activation and induce the lysis of cultured SKBR­3 and BT474 cells in the presence of unstimulated T lymphocytes. HER2/CD3 BsAb efficiently inhibited the growth of breast cancer tissue by activating and inducing the proliferation of tumor tissue infiltrating lymphocytes. Therefore, HER2/CD3 BsAb is a potent tool which may be a suitable candidate for the treatment of breast cancer.

High Phosphorylation Status of AKT/mTOR Signal in DESI2-Reduced Pancreatic Ductal Adenocarcinoma.

Desumoylating isopeptidase 2 (DESI2) is a recently identified protein with unclear functions. In this study, a total of 132 tissue samples of pancreatic ductal adenocarcinoma and 73 samples of pancreatic normal tissues were explored to assess DESI2 expression and its implications to AKT/mTOR signal. Immunohistochemistry showed DESI2 expression is significantly decreased in cancer tissues versus normal tissues, presenting lowest level in poorly differentiated cancer. Unlike DESI2, the key factors in AKT/mTOR pathway including p-AKT, mTOR, p-mTOR and p-P70S6K present high expression in pancreatic cancer. It is notable that p-mTOR is significantly increased in DESI2-lower cancer compared with DESI2-higher cancer, although mTOR presents no difference in the two groups. The relative p-mTOR/mTOR ratio is also significantly elevated in DESI2-lower cancer. Moreover, the samples whose p-AKT and p-mTOR scores both exceed the median are obviously increased in DESI2-lower cancer compared with DESI2-higher cancer. As a downstream molecule of AKT/mTOR pathway, p-P70S6K was found to display higher level in DESI2-lower pancreatic cancer. High phosphorylation status of those proteins in DESI2-reduced pancreatic cancer indicates that there is high activity of AKT/mTOR signal in condition of DESI2 reduction, which could provide clues to reveal the implications of DESI2 in carcinogenesis.

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma.

BACKGROUND: Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed. RESULTS: Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101. CONCLUSIONS: Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.

Implications of PPPDE1 expression in the distribution of plakoglobin and ß-catenin in pancreatic ductal adenocarcinoma.

Human PPPDE peptidase domain-containing protein 1 (PPPDE1) is a recently identified protein; however, its exact functions remain unclear. In our previous study, the PPPDE1 protein was found to be decreased in certain cancer tissues. In the present study, a total of 96 pancreatic ductal carcinoma tissue samples and 31 normal tissues samples were assessed to investigate the distribution of plakoglobin and ß-catenin under the conditions of various PPPDE1 expression levels by means of immunohistochemistry. Generally, the staining of PPPDE1 was strong in normal tissues, but weak in cancer tissues. Plakoglobin was mainly distributed along the membrane and cytoplasm border in normal cells, but was less evident in the membranes of cancer cells. In particular, a greater percentage of cells exhibited low membrane plakoglobin expression in cancer tissue with low PPPDE1 expression (PPPDE1-low cancer) compared with that in cancer tissue with high PPPDE1 expression (PPPDE1-high cancer). The distribution of ß-catenin in normal tissues was similar to that of plakoglobin. However, ß-catenin was peculiarly prone to invade nucleus in PPPDE1-low cancer compared with PPPDE1-high cancer. Our data suggested potential links between PPPDE1 expression and the distribution of plakoglobin and ß-catenin in pancreatic ductal adenocarcinoma, providing insights into the role of PPPDE1 in the progression of pancreatic cancer.

WNT16B from ovarian fibroblasts induces differentiation of regulatory T cells through ß-catenin signal in dendritic cells.

Treatment for cancer can induce a series of secreted factors into the tumor microenvironment, which can affect cancer progression. Wingless-type MMTV (mouse mammary tumor virus) integration site 16B (WNT16B) is a new member of the WNT family and has been reported to play growth-related roles in previous studies. In this study, we found WNT16B could be expressed and secreted into the microenvironment by human ovarian fibroblasts after DNA damage-associated treatment, including chemotherapy drugs and radiation. We also demonstrated that fibroblast-derived WNT16B could result in accumulation of ß-catenin in dendritic cells and secretion of interleukin-10 (IL-10) and transforming growth factor beta (TGF-ß), which contributed to the differentiation of regulatory T cells in a co-culture environment. These results shed light on the roles of WNT16B in immune regulation, especially in regard to cancer treatment.

The preparation of VEGFR1/CD3 bispecific antibody and its specific cytotoxicity against VEGFR1-positive breast cancer cells.

Bispecific antibody (BsAb) has been proved to be a very effective antitumor approach because of its distinctive advantages of immune-mediated cytotoxicity. To enhance the ability to recruit and activate T lymphocytes for tumor-specific killing, we constructed and prepared a recombinant human single-chain Fv bispecific antibody (BsAb), named VEGFR1/CD3 BsAb, targeting VEGFR1 and CD3. The VEGFR1/CD3 BsAb was expressed in CHO-K1 cells and purified by Ni-NTA affinity chromatography. The CD3 and VEGFR1-binding activity of VEGFR1/CD3 BsAb was confirmed by flow cytometry. T lymphocyte activation and proliferation induced by VEGFR1/CD3 BsAb were also demonstrated in vitro. Notably, our VEGFR1/CD3 BsAb presented a powerful and specific killing effect against VEGFR1-positive human breast cancer cell MDA-MB-231 and MDA-MB-435 through activating T lymphocyte at very low concentrations, indicating that it will be a valuable antibody drug for treatment of VEGFR1-positive cancers in the future.

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma

BACKGROUND: Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed. RESULTS: Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101. CONCLUSIONS: Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.

HOCl oxidation-modified CT26 cell vaccine inhibits colon tumor growth in a mouse model.

Despite progress in elucidating mechanisms associated with colorectal cancer and improvement of treatment methods, it remains a frequent cause of death worldwide. New and more effective therapies are therefore urgently needed. Recent studies have shown that immunogenicity of whole ovarian tumor cells and subsequent T cell response were potentiated by oxidation modification with hypochlorous acid (HOCl) in vitro and ex vivo. These results prompted us to investigate the protective antitumor response with an HOCl treated CT26 colorectal cancer cell vaccine in an in vivo mouse model. Administration of HOCl modified vaccine triggered robust antitumor immunity to autologous tumor cells in mice and prolonged survival period significantly. In addition, increased necrosis and apoptosis were found in tumor tissue from the oxidation group. Interestingly, ELISPOT assays showed that specific T cell responses were not elicited in response to the immunizing cellular antigen, in contrast to raising sera antibody titer and antibody binding activity shown by ELISA assay and flow cytometry. Further evaluation of the mechanisms underlying HOCl modified vaccine mediated humoral immunity highlighted the role of antibody-dependent cell-mediated cytotoxicity. These results combined with previous studies suggest that HOCl oxidation modified whole cell vaccine has wide applicability as a cancer vaccine because it can target both T cell- and B cell-specific responses. It may thus represent a promising approach for the immunotherapy of colorectal cancer.

A fully human CD19/CD3 bi-specific antibody triggers potent and specific cytotoxicity by unstimulated T lymphocytes against non-Hodgkin's lymphoma.

The use of a bi-specific antibody (BsAb) is an attractive and specific approach to cancer therapy. We have constructed a fully human recombinant single chain Fv BsAb against CD19 and CD3 that was an effective treatment in an animal model of non-Hodgkin's lymphoma (NHL). The CD19/CD3 BsAb was expressed in CHO cells and purified by Ni-column chromatography. Flow cytometry revealed that the CD19/CD3 BsAb specifically bound to both CD19 and CD3-positive cells. In vitro, the CD19/CD3 BsAb could stimulate T cell proliferation and induce the lysis of cultured Raji cells in the presence of unstimulated T lymphocytes. In vivo, the CD19/CD3 BsAb efficiently inhibited tumour growth in SCID mice of NHL, and the survival time of the mice was significantly prolonged. Therefore, our CD19/CD3 BsAb is a useful tool that could be a suitable candidate for treatment of NHL.

Expression of CD40 and growth-inhibitory activity of CD40 agonist in ovarian carcinoma cells.

The CD40 receptor is a member of the tumour necrosis factor receptor family and is widely expressed on various cell types. The antitumour activity of CD40 agonist antibody has been observed in B-cell-derived malignancies, but its activity on ovarian cancer remains unclear. However, in this paper, we first confirmed that the anti-CD40 agonist antibody could inhibit the growth of ovarian cancer cells and induce apoptosis. This study investigated the expression of CD40 by ovarian carcinoma tissues and cell lines, at the same time, we evaluated the effect of a recombinant soluble human CD40L (rshCD40L) and an anti-CD40 agonist antibody on cell growth and apoptosis. Flow cytometry and immunohistochemistry assay demonstrated that CD40 was expressed on ovarian carcinoma cell lines and primary ovarian carcinoma cells derived from ascites, as well as on ovarian carcinoma tissues. The growth inhibition of rshCD40L and the anti-CD40 agonist antibody on ovarian carcinoma cells was examined by MTT assay, and the proportion of apoptotic tumour cells was analysed by flow cytometry and Hoechst staining. Our study showed that CD40 was expressed on all ovarian carcinoma cell lines and was examined in 86.2% (162/188) of ovarian cancer tissue samples, but not in normal ovarian tissues (n = 20). Treatment with rshCD40L or anti-CD40 agonist antibody significantly inhibited ovarian carcinoma cell growth and induced apoptosis. Theses results suggest that CD40 is expressed on ovarian carcinoma cells, moreover, that rshCD40L and anti-CD40 agonist antibody have therapeutic potential to inhibit human ovarian cancer growth.

Comparative proteomic analysis of atrial appendages from rheumatic heart disease patients with sinus rhythm and atrial fibrillation.

Atrial fibrillation (AF) is the most common form of arrhythmia encountered in clinical practice, and contributes to cardiovascular morbidity and mortality. Despite significant advances in the understanding of the mechanisms associated with AF, the number of effective biomarkers and viable therapeutic targets remains relatively limited. In this study, 2-DE and MS/MS analysis was used to identify differentially expressed proteins in human atrial appendage tissues from patients with AF (n=4) compared to controls with sinus rhythm (SR; n=5). All subjects had rheumatic heart disease. Following 2-DE analysis, Coomassie Brilliant Blue staining and MS/MS identification, a total of 19 protein spots were found to be differentially expressed between the AF and SR groups. By cluster and metabolic/signaling pathway analysis, these proteins were divided into three major groups: proteins involved in the cytoskeleton and myofilament, energy metabolism associated proteins, and proteins associated with oxidative stress. The proteins identified in this study may enable a better understanding of the molecular mechanisms of AF, and may provide useful biomarkers and novel targets for drug development.

Polyclonal rabbit anti-murine plasmacytoma cell globulins induce myeloma cells apoptosis and inhibit tumour growth in mice.

Multiple myelomas (MMs) are etiologically heterogeneous and there are limited treatment options; indeed, current monoclonal antibody therapies have had limited success, so more effective antibodies are urgently needed. Polyclonal antibodies are a possible alternative because they target multiple antigens simultaneously. In this study, we produced polyclonal rabbit anti-murine plasmacytoma cell immunoglobulin (PAb) by immunizing rabbits with the murine plasmacytoma cell line MPC-11. The isolated PAb bound to plasma surface antigens in several MM cell lines, inhibited their proliferation as revealed by MTT assay, and induce apoptosis as indicated by flow cytometry, microscopic observation of apoptotic changes in morphology, and DNA fragmentation on agarose gels. The cytotoxicity of PAb on MPC-11 cell lines was both dose-dependent and time-dependent; PAb exerted a 50% inhibitory effect on MPC-11 cell viability at a concentration of 200 µg/ml in 48 h. Flow cytometry demonstrated that PAb treatment significantly increased the number of apoptotic cells (48.1%) compared with control IgG (8.3%). Apoptosis triggered by PAb was confirmed by activation of caspase-3, -8, and -9. Serial intravenous or intraperitoneal injections of PAb inhibited tumour growth and prolonged survival in mice bearing murine plasmacytoma, while TUNEL assay demonstrated that PAb induced statistically significant apoptosis (P < 0.05) compared to control treatments. We conclude that PAb is an effective agent for in vitro and in vivo induction of apoptosis in multiple myeloma and that exploratory clinical trials may be warranted.

Identification, characterization, and effects of Xenopus laevis PNAS-4 gene on embryonic development.

Apoptosis plays an important role in embryonic development. PNAS-4 has been demonstrated to induce apoptosis in several cancer cells. In this study, we cloned Xenopus laevis PNAS-4 (xPNAS-4), which is homologous to the human PNAS-4 gene. Bioinformatics analysis for PNAS-4 indicated that xPNAS-4 shared 87.6% identity with human PNAS-4 and 85.5% with mouse PNAS-4. The phylogenetic tree of PNAS-4 protein was also summarized. An analysis of cellular localization using an EGFP-fused protein demonstrated that xPNAS-4 was localized in the perinuclear region of the cytoplasm. RT-PCR analysis revealed that xPNAS-4, as a maternally expressed gene, was present in all stages of early embryo development. Whole-mount in situ hybridization showed that xPNAS-4 was mainly expressed in ectoderm and mesoderm. Furthermore, microinjection of xPNAS-4 mRNA in vivo caused developmental defects manifesting as a small eye phenotype in the Xenopous embryos, and as a small eye or one-eye phenotype in developing zebrafish embryos. In addition, embryos microinjected with xPNAS-4 antisense morpholino oligonucleotides (MOs) exhibited a failure of head development and shortened axis.

Altered protein-expressing profile in hPNAS4-induced apoptosis in A549 human lung adenocarcinoma cells.

Human PNAS4 (hPNAS4) is a recently identified pro-apoptosis gene, which is able to induce apoptosis in A549 human lung adenocarcinoma cells following its overexpression. In this work, we investigated the changes of protein profile in hPNAS4-induced apoptosis in A549 cells through proteomic strategy consisting of two-dimensional electrophoresis (2-DE) coupled with MALDI-Q-TOF mass spectrometry. A total of 20 different proteins with more than 3.0-fold change in expression, including 5 up-regulated and 15 down-regulated proteins were successfully identified by database search. The mRNA transcription levels of the different proteins were further examined by RT-PCT. Functional analyses showed these different proteins are involved in diverse biological processes including metabolism, proteolysis, signal transduction, apoptosis, and redox regulation. Two essential apoptosis-associated protein, annexin A1 and prothymosin alpha, were confirmed by Western blot and showed consistent changes with proteomic detection. Our data provide molecular evidence and possible associated pathway in hPNAS4-induced apoptosis through proteomic strategy, which should be contributed to further investigation on biological function of hPNAS4.

PNAS-4, a novel pro-apoptotic gene, can potentiate antineoplastic effects of cisplatin.

PURPOSE: PNAS-4, a novel pro-apoptotic gene activated during the early response to DNA damage, can inhibit proliferation via apoptosis when overexpressed in some tumor cells. The objectives of this study were to determine whether PNAS-4 could enhance apoptosis induced by cisplatin besides its induction of apoptosis, and to evaluate the usefulness of combined treatment with mouse PNAS-4 (mPNAS-4) gene therapy and low-dose cisplatin chemotherapy in the inhibition of tumor growth in colon carcinoma (CT26) and Lewis lung carcinoma (LL/2) murine models. METHODS: In this study, the in vitro growth-inhibitory and pro-apoptotic effects of PNAS-4 and/or cisplatin on CT26, LL/2, and SKOV3 cancer cells were assessed by MTT assay, flow cytometric analysis, DNA fragmentation, and morphological analysis, respectively. The in vivo antitumor activity of combined treatment with mPNAS-4 gene therapy and low-dose cisplatin were evaluated in the inhibition of tumor growth in colon carcinoma (CT26) and Lewis lung carcinoma (LL/2) murine models. Tumor volume and survival time were observed. Induction of apoptosis was also assessed in tumor tissues. RESULTS: In vitro, PNAS-4 inhibited proliferation of colon carcinoma (CT26), Lewis lung carcinoma (LL/2) and human ovarian cancer (SKOV3) cell lines via apoptosis, and significantly enhanced the apoptosis of CT26, LL/2, and SKOV3 cells induced by cisplatin. In vivo systemic administration of expression plasmid encoding mPNAS-4 (pcDNA3.1-mPS) and cisplatin, significantly decreased tumor growth through increased tumor cell apoptosis compared to treatment with mPNAS-4 or cisplatin alone. CONCLUSIONS: Our data suggests that the combined treatment with mPNAS-4 plus cisplatin may augment the induction of apoptosis in tumor cells in vitro and in vivo, and that the augmented antitumor activity in vivo may result from the increased induction of apoptosis. The present study may provide a novel way to augment the antitumor efficacy of cytotoxic chemotherapy.

Identification of ATP synthase beta subunit (ATPB) on the cell surface as a non-small cell lung cancer (NSCLC) associated antigen.

BACKGROUND: Antibody-based immunotherapy has achieved some success for cancer. But the main problem is that only a few tumor-associated antigens or therapeutic targets have been known to us so far. It is essential to identify more immunogenic antigens (especially cellular membrane markers) for tumor diagnosis and therapy. METHODS: The membrane proteins of lung adenocarcinoma cell line A549 were used to immunize the BALB/c mice. A monoclonal antibody 4E7 (McAb4E7) was produced with hybridoma technique. MTT cell proliferation assay was carried out to evaluate the inhibitory effect of McAb4E7 on A549 cells. Flow cytometric assay, immunohistochemistry, western blot and proteomic technologies based on 2-DE and mass spectrometry were employed to detect and identify the corresponding antigen of McAb4E7. RESULTS: The monoclonal antibody 4E7 (McAb4E7) specific against A549 cells was produced, which exhibited inhibitory effect on the proliferation of A549 cells. By the proteomic technologies, we identified that ATP synthase beta subunit (ATPB) was the corresponding antigen of McAb4E7. Then, flow cytometric analysis demonstrated the localization of the targeting antigen of McAb4E7 was on the A549 cells surface. Furthermore, immunohistochemistry showed that the antigen of McAb4E7 mainly aberrantly expressed in tumor cellular membrane in non-small cell lung cancer (NSCLC), but not in small cell lung cancer (SCLC). The rate of ectopic expressed ATPB in the cellular membrane in lung adenocarcinoma, squamous carcinoma and their adjacent nontumourous lung tissues was 71.88%, 66.67% and 25.81% respectively. CONCLUSION: In the present study, we identified that the ectopic ATPB in tumor cellular membrane was the non-small cell lung cancer (NSCLC) associated antigen. ATPB may be a potential biomarker and therapeutic target for the immunotherapy of NSCLC.

Comparative plasma membrane-associated proteomics of immortalized human hepatocytes.

This work was initiated with the purpose of purifying and identifying differentially expressed plasma membrane-associated proteins between human liver cancer cell line HepG2 and normal liver cell line L02. The combined strategy of sucrose density gradient centrifugation and subsequent phase partition was applied to obtain high-purity proteins of plasma membrane. Two-dimensional gel electrophoresis revealed the differential protein profile between the two cell lines. A total of 13 plasma membrane-associated proteins containing 10 up-regulated proteins and three down-regulated proteins in HepG2 cells were successfully identified by MALDI-Q-TOF mass spectrometry; they participate in multiple biological functions such as adhesion, proliferation, apoptosis, and signal transduction. The identified proteins could provide helpful reference in clinical investigations on potential candidates for diagnosis and therapy of liver cancer.

[Association between serum lipoprotein-cholesterol levels and HDL subpopulations].

OBJECTIVE: To investigate the influence of serum HDL-C or LDL-C levels on the components of serum HDL subpopulations. METHODS: Apolipoprotein (apo) A-I contents of serum HDL subpopulations in 292 subjects were determined by two-dimensional gel electrophoresis in conjunction with immunodection. RESULTS: With the decrease of serum HDL-C levels, the apoA-I contents of pre beta1-HDL and HDL3b increased and were significantly higher (P<0.01; P<0.05) in the low HDL-C group than in the high HDL-C group. But on the contrary, with the decrease of serum HDL-C levels, the apoA-I contents of HDL2b and HDL2a decreased and were significantly lower (P<0.01) in the middle and low HDL-C groups than in the high HDL-C group. With the increase of serum LDL-C levels, the apoA-I contents of pre beta1-HDL, HDL3C and HDL3b increased; the apoA-I contents of pre beta1-HDL and HDL3b were significantly higher in the high LDL-C group (P<0.05) and very high LDL-C group (P<0.01), and those of HDL3C were also significantly higher in the very high LDL-C group (P<0.01). But on the contrary, with the increase of LDL-C levels, the apoA-I contents of HDL2b decreased and were significantly lower in the high LDL-C group (P<0.05) and very high LDL-C group (P<0.01) when compared with the apoA-I contents of the desirable LDL-C group. CONCLUSION: With the decrease of serum HDL-C levels or increase of serum LDL-C levels, the small-sized HDL increased and the large-sized HDL decreased; furthermore, the HDL-C levels were more closely related to the components of the large-sized HDL (HDL2a, HDL2b).