Cytokeratin 18-mediated disorganization of intermediate filaments is induced by degradation of plectin in human liver cells.

Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork that helps maintain the uniform size and shape of cells. As cells of hepatocellular carcinoma are morphologically different from healthy human hepatocytes, we hypothesized that plectin deficiency and cytoskeletal disorganization underlies this pleomorphic transformation. To test this hypothesis we induced apoptosis as the most accessible pathway for creating plectin deficiency status in vivo. We analyzed expression levels and organization of plectin and other cytoskeletal elements, including intermediate filaments, microfilaments, and microtubules, after staurosporine-induced apoptosis in human Chang liver cells. The results revealed the expression of plectin and cytokeratin 18 were downregulated in hepatocellular carcinoma tissues in vivo. The expression of actin and tubulin, however, were not altered. In vitro analysis indicated that plectin and cytokeratin 18 were cleaved following staurosporine-treatment of human Chang liver cells. Time course experiments revealed that plectin was cleaved 2h earlier than cytokeratin 18. The organization of plectin and cytokeratin 18 networks collapsed after staurosporine-treatment. Conclusively, degradation of plectin induced by staurosporine-treatment in liver cells resulted in cytoskeleton disruption and induced morphological changes in these cells by affecting the expression and organization of cytokeratin 18.

Plectin deficiency on cytoskeletal disorganization and transformation of human liver cells in vitro.

Plectin is a versatile cytoplasmic cross-linking protein that connects intermediate filaments to microfilaments, microtubules, and membrane adhesion sites. The cross-linking functions of plectin help organize the cytoskeleton into a stable meshwork important for maintaining uniformity in cell size and shape. As cells of hepatocellular carcinoma are morphologically different from normal human hepatocytes, we hypothesized that altered plectin expression and cytoskeletal organization underlies this pleomorphic transformation. To test this hypothesis, we analyzed expression levels and organization of all cytoskeletal elements, including intermediate filaments, microfilaments, and microtubules, after plectin knockdown in human Chang liver cells. We found that expression of cytokeratin 18, but not actin or tubulin, was downregulated by suppression of plectin protein. Furthermore, cytokeratin networks were partially collapsed and actin-rich stress fibers were increased. The organization of microtubule networks, by contrast, was unaltered. These findings support our hypothesis that, via effects on cytoskeletal organization, plectin deficiency might play an important role in the transformation of human liver cells.

Synemin down-regulation in human hepatocellular carcinoma does not destabilize cytoskeletons in vivo.

Synemin is a large intermediate filament protein that has been identified in all types of muscle cells. It plays a role in human muscle diseases; however, the role of synemin in tumor cell transformation has rarely been investigated. Because hepatocellular carcinoma cells are morphologically different from normal human hepatocytes, we hypothesized that altered synemin expression and cytoskeletal disorganization might underlie this pleomorphic transformation. To test this hypothesis, we studied synemin expression in hepatocellular carcinoma and liver tissues by immunohistochemistry and immunoblotting. In addition, we analyzed the expression level and organization of all cytoskeletal elements after synemin knock-down in human Chang liver cells. Previously we found that plectin knock-down in human Chang liver cells causes a reduction in cytokeratin 18 expression with effects on intermediate filament disorganization and altered cellular morphology. In this study we also compared the effects of synemin knock-down and plectin knock-down on the cytoskeleton expression and organization. The results revealed that synemin expression was down-regulated in human hepatocellular carcinoma compared with normal liver, which is similar to the plectin expression. Surprisingly, the expression of cytoskeletal elements (cytokeratin 18, actin and tubulin) was not influenced by synemin knock-down in human Chang liver cells. The organization of cytoskeletal networks was also unaltered after synemin knock-down. In conclusion, both plectin and synemin are down-regulated in human hepatocellular carcinoma in vivo and transformed human liver cell in vitro. However, the mechanism of cell transformation caused by synemin knock-down is different from that of plectin knock-down. Plectin, but not synemin, knock-down provoked liver cell transformation via suppressing cytokeratin 18 expression and disrupting intermediate filament networks. Synemin knock-down did not influence the cytoskeleton expression and organization of human Chang liver cells.

Co-treating with arecoline and 4-nitroquinoline 1-oxide to establish a mouse model mimicking oral tumorigenesis.

The aim of this study was to establish an effective mouse model of oral cancer and to use this model to identify potential markers of oral tumor progression. C57BL/6JNarl mice were treated with arecoline, 4-nitroquinoline 1-oxide (4-NQO), or both arecoline and 4-NQO in high and low doses for 8 weeks to induce oral tumor. The induced oral lesions were observed for 20 weeks to assess the efficiency of cancer induction and survival rate of the mice. In addition, two target proteins that are frequently overexpressed during tongue cancer tumorigenesis, alphaB-crystallin and Hsp27, were examined by immunohistochemical analysis. In mice exposed to 4-NQO (200 microg/mL) and arecoline (500 microg/mL), the tongue lesions showed evidence of hyperplasia, papilloma, dysplasia, and carcinoma, and the lesions were pathologically similar to those lesions in human oral cancer. The tongue tumor incidence rate was 100% in mice exposed to concomitant 4-NQO (200 microg/mL) and arecoline (500 microg/mL) treatment, 57% in mice exposed to 4-NQO only, and 0% in mice exposed to arecoline only. Immunohistochemical analysis demonstrated that, consistent with human studies, alphaB-crystallin and Hsp27 were upregulated in murine oral tumors. In conclusion, we have established a powerful animal model that enables the study of the promoting effects of arecoline on tongue tumorigenesis. Data subsequently attained from this mouse model support a role for alphaB-crystallin and Hsp27 as clinical markers for tumor progression.

Degradation of plectin with modulation of cytokeratin 18 in human liver cells during staurosporine-induced apoptosis.

BACKGROUND: Hepatoma cells are morphologically different from those of the normal liver. Intermediate filaments (IFs) are important in building the cellular architecture and maintaining the outline of cells. Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork, which can maintain the uniform size and shape of hepatocytes. Apoptosis might be the most possible pathway for creating plectin deficiency in the in vivo state. MATERIALS AND METHODS: Apoptosis was induced by staurosporine (STS) treatment in liver cells. The protein expression of cytokeratin 18 (CK18) and plectin as well as the morphology of the liver cells and the distribution of CK18 and plectin in the cells was studied after STS treatment. RESULTS: Plectin was cleaved in the liver cells during apoptosis and CK18 was modulated. Morphological changes were observed in the liver cells. CONCLUSION: By affecting the organization of IFs, plectin might play an important role in the pleomorphism of hepatoma cells and even the tumorigenesis of hepatoma.

Possible relation between histone 3 and cytokeratin 18 in human hepatocellular carcinoma.

BACKGROUND: Previously we found some low molecular weight proteins identified as histone in hepatocelluar carcinoma. Our objective was to clarify whether the coimmunoprecipitation of histone and cytokeratin 18 was an artifact or not. MATERIALS AND METHODS: Histone 3 and cytokeratin 18 were investigated in three cases of human hepatocellular carcinoma and one case of normal liver tissue. Nuclei of the tissues were isolated; the proteins inside the nuclei were analyzed by Western blot. RESULTS: The results revealed histone was co-immunoprecipitated with cytokeratin 18 in hepatocellular carcinoma. It was speculated that modulation of the cytoskeleton in human hepatocellular carcinoma might disturb the organization of the nucleoskeleton. The unstable nucleoskeleton might further cause instability and fragility of nuclei, thus possibly exposing the histone and co-immunoprecipitating it with cytokeratin 18. CONCLUSION: The evidence might indicate that expression of histone 3 was highly related to modulation of cytokeratin 18 and might play an important role in tumorigenesis of hepatocellular carcinoma.

The influence of plectin deficiency on stability of cytokeratin18 in hepatocellular carcinoma.

Intermediate filaments are important in building the cellular architecture. Previously we found cytokeratin18 was modulated in human hepatocellular carcinoma. Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork, which can maintain the uniform size and shape of hepatocytes. Because the cells of hepatocellular carcinoma were morphologically different from the hepatocytes, we speculated that expression of plectin and organization of intermediate filament might play roles in the pleomorphism of hepatocellular carcinoma cells. In this paper, we studied the plectin expression of hepatocellular carcinoma and liver tissues by immunohistochemistry and immunoblot. The results revealed that plectin was deficient and cytokeratin18 was modulated in hepatocellular carcinoma. Furthermore, we knockdown the plectin mRNA in Chang cells, the result revealed the plectin was deficient and the organization of cytokeratin18 was altered. Conclusively, this study offers a hypothesis that plectin deficient might play an important role in the tumorigenesis of hepatocellular carcinoma.

Pleomorphism of cancer cells with the expression of plectin and concept of filament bundles in human hepatocellular carcinoma.

Intermediate filaments are important in building the architecture of liver cells and are proposed to interact with other cellular components. Among intermediate filament associated proteins, plectin is a versatile cytoskeletal linkage protein which has been shown to interact with a variety of cytoskeletal structures. Intermediate filament and plectin might play some roles in tumorigenesis of human hepatocellular carcinoma since cells of hepatocellular carcinoma were morphologically different from normal liver. Plectin exhibited wide distribution spectrum among various tissues, however, it was poorly investigated in human liver and hepatoma tissues. In this paper, we studied the plectin expression in 18 cases of human hepatocellular carcinoma and normal hepatocytes by immunohistochemistry. The results revealed that plectin expression was deficient in human hepatocellular carcinoma and was probably through post-translational modification. Many 0.4 to 0.8 microm-thick keratin bundles were found in intermediate filament extracts of liver and hepatoma tissues. These bundles were greater in diameter about 40 to 80 times of single intermediate filament. We speculated that intermediate filament organized into "filament bundles" to maintain the shape of normal cells. In cancer cells, plectin was deficient and the irregularly loosened filament bundles could cause pleomorphism of cancer cells.

Immunohistochemical profiles of Mallory body by a panel of anti-cytokeratin antibodies.

Mallory bodies (MBs) are hyaline inclusions found in a variety of liver diseases. Because the major components of MBs are cytokeratins (CKs), CK profiles of MBs were examined immunohistochemically in 37 autopsied liver specimens (including a variety of nontumor pathological livers and hepatocellular carcinomas), using 13 antibodies against specific CKs: 34betaB4 (CK 1), OV-TL12/30 (CK 7), 34betaH11 (CK 8), LHP1 (CK 10), KS-1A3 (CK 13), LL002 (CK 14), LHK15 (CK 15), LL025 (CK 16), E3 (CK 17), DC10 (CK 18), b170 (CK 19), Ks20.8 (CK 20), and 34betaE12 (CKs 1, 5, 10, and 11). Positive staining rates of MBs in 37 cases were as follows: CK 8, 100% (37/37); CK 18, 100% (37/37); CK 19, 57% (21/37). CK 7, 49% (18/37); CK 20, 35% (13/37); CK 1, CK 5, CK 10, CK 11, CK 13, CK 14, CK 15, CK 16, and CK 17 were all negative in MBs. CK expression patterns in MBs found in tumor or non-tumor hepatocytes was basically similar. Thus, all present MBs were composed of similar material with common antigenic determinants, regardless of underlying disease; in particular, they consistently contained CK 8 and CK 18, and frequently contained CK 7, CK 19, and CK 20.

Effects of diode 808 nm GaAlAs low-power laser irradiation on inhibition of the proliferation of human hepatoma cells in vitro and their possible mechanism.

Low-power laser irradiation (LPLI) has come into a wide range of use in medical field. Considering basic research, LPLI can enhance DNA synthesis and increases proliferation rate of human cells. But only a few data about the effects of LPLI on human liver or hepatoma cells are available. The cytoskeleton plays important roles in cell function and therefore is implicated in the pathogenesis of many human liver diseases, including malignant tumors. In our previous study, we found the stability of cytokeratin molecules in human hepatocytes was related to the intact microtubule network that was influenced by colchicine. In this study, we are going to search the effect of LPLI on proliferation of human hepatoma cell line HepG2 and J-5 cells. In addition, the stability of cytokeratin and synemin (one of the intermediate filament-associated proteins) were analyzed under the action of LPLI to evaluate the possible mechanism of LPLI effects on proliferation of human hepatoma cells. In experiment, HepG2 and J-5 cells were cultured in 24-well plate for 24 hours. After irradiation by 130 mW diode 808 nm GaAlAs continue wave laser in different time intervals, the cell numbers were counted. Western blot and immunofluorescent staining examined the expression and distribution of PCNA, cytokeratin and synemin. The cell number counting and PCNA expression were evaluated to determine the proliferation. The organization and expression of cytokeratin and synemin were studied to identify the stability of cytoskeleton affected by LPLI. The results revealed that proliferation of HepG2 and J-5 cells was inhibited by LPLI since the cell number and PCNA expression was reduced. Maximal effect was achieved with 90 and 120 seconds of exposure time (of energy density 5.85 J/cm2 and 7.8 J/cm2, respectively) for HepG2 and J-5, respectively. The decreased ratio of cell number by this dose of irradiation was 72% and 66% in HepG2 and J-5 cells, respectively. Besides that, the architecture of intermediate filaments in these cells was disorganized by laser irradiation. The expression of intermediate filament-associated protein, synemin, was also reduced. Two significant findings are raised in this study: (1) Diode 808 nm GaAlAs continuous wave laser has an inhibitory effect on the proliferation of human hepatoma cells line HepG2 and J-5. (2) The mechanism of inhibition might be due to down-regulation of synemin expression and alteration of cytokeratin organization that was caused by laser irradiation.

An early evaluation of malignant tendency with plectin expression in human colorectal adenoma and adenocarcinoma.

Tumor cells are morphologically different from normal cells. In this situation, we proposed that plectin, one of the intermediate filament associated proteins, might play some special roles in the tumorigenesis. Plectin exhibits a wide distribution spectrum among various tissues; however, it is scarcely investigated in tumor tissues including colorectal adenocarcinoma. In this study, we searched the plectin expression in 25 cases of colorectal adenocarcinoma and 10 cases of tubular adenoma with focal adenocarcinoma by immunohistochemical method. The results reveal that plectin is up-regulated in colorectal adenocarcinoma as well as in bizarre glands and locally invasive tumor nests in tubular adenoma, compared with normal colorectal mucosa. Over-expression of plectin in locally invasive tumor nests might help us in diagnosis of distinguish microinvasive foci from normal glands. The bizarre glands in adenoma might be precancerous lesions since the expression of plectin in them was identical to that of adenocarcinoma but in contrast to that of normal glands. The overexpression of plectin might affect the organization of cytoskeleton, which might further cause tumorigenesis and morphological change of colorectal epithelial cells.

Altered synemin could affect the organization of intermediate filament in human hepatocellular carcinoma.

Cells of human hepatocellular carcinoma (HCC) were morphologically different from those of normal liver. Intermediate filaments are important in building the architecture of liver cells and are proposed to interact with other cellular components. Synemin is one of intermediate filament associated proteins which can link between intermediate filament and other cytoskeletal structures. It was suggested that synemin might play some roles in tumorigenesis of hepatocellular cancinoma. In this study, we searched the synemin expression in 18 human HCCs by immunohistochemistry. The results revealed that synemin was modulated nearly in all human HCC cases. Many 0.4-0.8 microm-thick bundles were found in the IF extracts of liver and HCCs. These bundles were greater in diameter about 40 to 80 times of single IF. We speculated that the IFs were organized into the "filament bundles" to support the normal shape of the cells. That was, normal cell needed numerous synemin to lock the individual IF into stable "filament bundles". In cancer cells, the synemin was altered and this might lead to a loosened change of filament bundles, and the cancer cells would become pleomorphic.

Prepubertal resveratrol exposure accelerates N-methyl-N-nitrosourea-induced mammary carcinoma in female Sprague-Dawley rats.

The major object of this study was to characterize the effect of prepubertal trans-3,4',5-trihydroxystilbene (resveratrol) exposure on N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis in female Sprague-Dawley rats. Prepubertal rats (15 to 19 days of age) were treated daily with either 10 or 100 mg/kg resveratrol for 5 days, and were compared with resveratrol-untreated animals (30 rats in each group). Six rats in each group were autopsied at 49 days of age, and their growth was evaluated. All remaining rats were given 50 mg/kg MNU, followed by monitoring for occurrence of mammary carcinoma. A dose of 100 mg/kg (but not 10 mg/kg) resveratrol significantly increased incidence of rat with mammary carcinomas > or =1 cm and multiplicity (all histologically detected mammary carcinomas per rat), but did not affect latency, compared with untreated controls. Resveratrol did not affect body weight increase, but 100 mg/kg resveratrol caused slightly earlier vaginal opening. Although all rats cycled, resveratrol-treated animals exhibited significantly increased irregularity of estrous cycle, spending more time in the estrus phase. Thus, short resveratrol treatment of prepubertal female rats affected endocrine function, and accelerated development of MNU-induced mammary carcinomas.

Effect of prenatal and prepubertal genistein exposure on N-methyl-N-nitrosourea-induced mammary tumorigenesis in female Sprague-Dawley rats.

BACKGROUND: The effect of prenatal and prepubertal genistein exposure on the development of chemically-induced mammary carcinomas in rat was investigated. MATERIALS AND METHODS: Genistein was subcutaneously (s.c.) injected daily, from gestational days 15 to 19, into pregnant Sprague-Dawley rats at 0, 1.5 or 30 mg/kg/day. Female offspring of mothers not exposed to genistein during pregnancy received daily s.c. injection, from prepubertal days 15 to 19, at a dose of 1.5 or 30 mg/kg/day. At 28 days of age, 6 female offspring from each group were sacrificed to observe the influence of genistein and the remaining rats were injected intraperitoneally with 50 mg/kg N-methyl-N-nitrosourea (MNU). Rats injected with MNU were sacrificed at 26 weeks of age or when their largest mammary tumor was > or = 1 cm in size. RESULTS: At the time when MNU was administered, the different groups had comparable mammary gland development; genistein-treated and -untreated rats had similar numbers of terminal end buds (TEBs) at the periphery of the mammary glandular tree. However, estrogen receptor alpha (ER alpha)- and progesterone receptor (PgR)-positive cells, p63-positive cells and proliferating cell nuclear antigen (PCNA)-labeling index were lower in genistein-exposed TEBs. Although tumor multiplicity and latency were not significant, prenatal or prepubertal genistein exposure, at low or high dosage, tended to suppress the incidence of mammary carcinomas > or = 1 cm; suppression was significant in the prepubertal low-dose group. CONCLUSION: The majority of the mammary carcinomas in the present study were hormone-dependent. The present findings suggest that administration of genistein in the perinatal period has protective effects against MNU-induced mammary carcinoma in Sprague-Dawley rats, via reduction of levels of ER alpha- and/or PgR-positive cells (presumed progenitor cells of mammary carcinomas), p63-positive mammary progenitor/stem cells (involved in cell renewal) and PCNA-positive cells (necessary for cell proliferation).

Prepubertal zearalenone exposure suppresses N-methyl-N-nitrosourea-induced mammary tumorigenesis but causes severe endocrine disruption in female Sprague-Dawley rats.

The effect of prepubertal exposure to zearalenone, an estrogenic mycotoxin, on N-methyl-N-nitrosourea (MNU)-induced mammary tumorigenesis and its influence on reproductive organs were examined in female Sprague-Dawley rats. Prepubertal rats were treated daily with either 0.1 or 10 mg/kg body weight of zearalenone between 15 and 19 days of age and compared with zearalenone-untreated animals (30 rats in each group). Six rats in each group were autopsied at 28 days of age, and their growth was evaluated. All remaining rats were given 50 mg/kg body weight MNU at 28 days of age and followed by monitoring for occurrence of mammary tumors > or =1 cm in diameter. Zearalenone did not affect body weight increase, and mammary glands showed similar development at 28 days of age (time at carcinogen administration). Both low- and high-dose zearalenone treatment significantly reduced incidence of mammary tumors > or =1 cm in diameter but did not influence latency (time between MNU administration and harvest of mammary tumor > or =1 cm in diameter) compared with untreated controls. Zearalenone dose dependently suppressed the number of histologically detected tumors (carcinomas) and multiplicity; the suppression was significant with high-dose treatment. However, high-dose treatment caused significantly earlier vaginal opening, both low- and high-dose treatment significantly caused irregularity of estrous cycle (persistent estrus or prolonged diestrus) at 8 to 10 wk of age, and zearalenone dose dependently increased the number of anovulatory rats (ovaries without newly formed corpora lutea) at 37 wk of age. Thus, short-duration zearalenone treatment in the prepubertal period suppressed subsequent mammary cancer occurrence but also severely damaged ovarian functions. This suggests that ingestion of foods containing zearalenone in the infantile period can have dramatic effects in later life.

HCC CKs are altered histones during tumor transformation in hepatoma.

The stability of cytokeratin (CK) protein during tumor transformation in human hepatocellular carcinoma (HCC) was studied with molecular approach previously. The results demonstrated that the CK was modulated in human HCC. Besides this, three low molecular weight CK molecules (named HCC CK) were found. It indicated that these HCC CKs are undergone modulation from human hepatocyte CK18. However, there were many differences between the CK18 and HCC CK. First, the antigenecity of HCC CK had been changed since they could not be recognized by CAM5.2 antibody on Western blot. Second, the sequences of N-terminal residues of HCC CK were matched with those of the N-terminal residues of human histone. In this study, we confirmed that the HCC CK was actually to be histones because they reacted to anti-histone antibody on Western blot. Furthermore, we found that the histones of human HCC had been changed during the process of tumor transformation since they could be co-immunoprecipitated with CK18 and could be detected by Western blot while this phenomenon did not happen in the normal liver tissue. We also found that not all histones change in human HCC. Only H3 was detected on Western blot while H1, H2A, H2B, and H4 were not detected in human HCCs.