Regulation of spermatogenesis by paracrine/autocrine testicular factors

Spermatogenesis is a complex process regulated by endocrine and testicular paracrine/autocrine factors. Gonadotropins are involved in the regulation of several testicular paracrine factors, mainly of the IL-1 family and testicular hormones. Testicular cytokines and growth factors (such as IL-1, IL-6, TNF, IFN-γ, LIF and SCF) were shown to affect both the germ cell proliferation and the Leydig and Sertoli cells functions and secretion. Cytokines and growth factors are produced by immune cells and in the interstitial and seminiferous tubular compartments by various testicular cells, including Sertoli, Leydig, peritubular cells, spermatogonia, differentiated spermatogonia and even spermatozoa. Corresponding cytokine and growth factor receptors were demonstrated on some of the testicular cells. These cytokines also control the secretion of the gonadotropins and testosterone in the testis. Under pathological conditions the levels of pro-inflammatory cytokines are increased and negatively affected spermatogenesis. Thus, the expression levels and the mechanisms involved in the regulation of testicular paracrine/autocrine factors should be considered in future therapeutic strategies for male infertility.

Gastrin as an autocrine growth factor in colorectal carcinoma:implications for therapy

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SIMULTANEOUS OVER-EXPRESSION OF INSULIN-LIKE GROWTH FACTOR- Ⅱ (IGF- Ⅱ ) AND IGF- Ⅱ RECEPTOR(IGF- Ⅱ R) GENES IN HUMAN PRIMARY CANCER-IMPLICATION OF AUTOCRINE AND PARACRINE MECHANISM IN AUTONOMOUS GROWTH OF HEPATIC CANCER

This is first report about the simultaneous over-expression of both Insulin-like growth factor (IGF- I ) and its receptor (IGF- I R) at mRNA level in human primary hepatic Cancer (PHC). In 10 PHC samples from China, IGF-I and IGF- I R were both over-expressed, whereas only a background signal was detected in normal liver. In 5 pairs of PHC and its non- tumorous adjacent liver tissues from South Africa, IGF- I and IGF- I R were also over-expressed in PHC. mRNA expression of IGF- I in all 5 cases and IGF- I R in 4 of 5 cases were higher in cancer than non- tumorous adjacent liver tissues. These results strongly implicate that an autocrine and/ or paracrine mechanism might be Involved in formation and progression of PHC.

PARTIAL PURIFICATION AND CHARACTERIZATION OF AN AUTOCRINE T SUPPRESSOR FACTOR FROM MURINE LEUKEMIA

The leukemia-associated autoinhibitor (LAI-615) derived from murine leukemia L7811 has been investigated intensively in our laboratory. In the following experiments, the partial purification of LA I-615 has been carried out in addition to the observation of phenotype variations of L7811 leuke-mic cells. The factor was purified over 1306-fold by sequential fractionation with Sephadex G-150 gel filtration, DEAE-cellulose ion exchange chromato-graphy, and Mono Q-fast protein liquid chromato-graphy. The molecular weight of LAI-615 was 68,000 as estimated by gel filtration. LAI-615 was a protein but not glycosylated, and it was suggested LAI-615 be secreted in an autocrine manner. Im-munocytochemical staining showed that the expression of Lyt2 phenotype of L7811 leukemic cells was often coincident with the secretion of LAI-615. Moreover, the physicochemical characteristics of LAI-615 was similar to that of T suppressor factor. Thus it is concluded that LAI-615 may be one of TsF-like factors.

Autocrine Production of Interleukin-6: A Mechanism of Interleukin-6 Independence in Dexamethasone-Resistant 7TD1 Murine Myeloma Cells

Several factors could contribute to proliferation of multiple myeloma (MM) cells independent of interleukin-6 (IL6) in the later stages of the disease. Our previous studies established a dexamethasone-resistant 7TD1 cell line (7TD1-Dxm) and have shown that one mechanism of resistance to dexamethasone is due to inhibition of cytochrome c release. We have also observed that 7TD1-Dxm cells proliferate independently of externally-added IL6. This study therefore aimed to elucidate the mechanisms responsible for IL6-independent proliferation in 7TD1-Dxm cells. Our results indicated that 7TD1-Dxm cells produced IL6 in an autocrine fashion. We have observed that dexamethasone-resistant 7TD1 cells become dexamethasone-resistant and IL6-independent for proliferation concomitantly. This strongly suggests that production of IL6 by 7TD1-Dxm cells may play an important role in the development of dexamethasone resistance. Consequently, further investigation of the molecular mechanisms responsible for IL6 production may be helpful in delineating the mechanisms leading to dexamethasone resistance.

MORPHOLOGICAL EVIDENCE FOR THE AUTOCRINE HYPOTHESIS

Autocrine secretion is a concept which emerges in the search for the molecular basis of malignant tumour. Tumour cells can produce endogenous polypeptide growth factors which act on their producer cells via functional external receptors. This process has been termed 'autocrine secretion'. Autocrine hypothesis explains the malignant growth of tumour cells satisfactorily. Transforming growth factor-α (TGF-α)is one of the typical autocrine growth factors. TGF-α can induce the reversible phenotypic transformation of normal rat kidney cells in the presence of other growth factors. This suggests that TGF-α may play an im-

Effect of Fuzhenghuayu decoction on vascular endothelial growth factor secretion in hepatic stellate cells

Objective: To investigate the effect of Fuzhenghuayu decoction on autocrine activation of hepatic stellate cell (HSC). Methods: The drug serum containing Fuzhenghuayu decoction was collected from normal rats, and cul- tured with activated HSC in vitro. The conditioned medium from the drug serum treated HSC was added to primary cultured quiescent HSC. Cell prolifera- tion was assayed by tetrazolium colorimetric test, and the contents of type Ⅰ collagen and vascular endo- thelial growth factor (VEGF) in the supernatant were measured with ELISA. Results: The conditioned medium from activated HSC could stimulate the quiescent HSC proliferation and type Ⅰ collagen secretion. The drug serum inhibi- ted this stimulating action and VEGF secretion from the activated HSC. Conclusion: Fuzhenghuayu decoction acts effectively against the autocrine activation pathway of HSC. The mechanism may be associated with the inhibition of the secretion of VEGF by activated HSC.

Hepatopoietin Cn suppresses apoptosis of human hepatocellular carcinoma cells by up-regulating myeloid cell leukemia-1

AIM:To investigate the role of hepatopoietin Cn(HPPCn) in apoptosis of hepatocellular carcinoma(HCC)cells and its mechanism. METHODS:Two human HCC cell lines,SMMC7721 and HepG2,were used in this study.Immunostaining, Western blotting and enzyme linked immunosorbent assay were conducted to identify the expression of HPPCn and the existence of an autocrine loop of HPPCn/ HPPCn receptor in SMMC7721 and HepG2.Apoptotic cells were detected using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide.RESULTS:The HPPCn was highly expressed in human HCC cells and secreted into culture medium(CM). FITC-labeled recombinant human protein(rhHPPCn) could specifically bind to its receptor on HepaG2 cells. Treatment with 400 ng/mL rhHPPCn dramatically increased the viability of HCC-derived cells from 48.1% and 36.9%to 85.6%and 88.4%,respectively(P< 0.05).HPPCn silenced by small-interfering RNA reduced the expression and secretion of HPPCn and increased the apoptosis induced by trichostatin A.Additionally, HPPCn could up-regulate the expression of myeloid cell leukemia-1(Mcl-1)in HCC cells via mitogen-activated protein kinase(MAPK)and sphingosine kinase-1. CONCLUSION:HPPCn is a novel hepatic growth factor that can be secreted to CM and suppresses apoptosis of HCC cells by up-regulating Mcl-1 expression.

CO-EXPRESSION OF MACROPHAGE COLONY-STIMULATING FACTOR WITH ITS RECEPTOR IN HUMAN HEPATOMA CELLS AND ITS POTENTIAL ROLES

Objective: To investigate the potential role of macrophage colony-stimulating factor (M-CSF) and macrophage colony-stimulating factor receptor (M-CSF-R) on the growth of human hepatoma cells. Methods: Specimens of different origin, including tissues of human hepatocellular carcinoma (HCC), human fetal liver (FL) and normal liver (NL), the hepatoma cell lines, as well as the peripheral blood mononuclear cells (PBMC) from patients with HCC or liver metastatic tumor (LMT), were used to detect the expression levels of M-CSF and M-CSF-R by ABC immunohistochemistry staining and reverse transcription polymerase chain reaction methods the expression levels of M-CSF and M-CSF-R. Influence of monoclonal antibody against M-CSF (B5) or M-CSF-R (RE2) on proliferation ability of hepatoma cell linesin vitro was also studied. Results: The results showed that hepatoma tissues produced elevated levels of both M-CSF and M-CSF-R compared with those of fetal liver (P<0.001). The M-CSF/M-CSF-R expression levels of PBMC from hepatoma patients were higher than those of LMT patients (P<0.01,P<0.05) and the normal people (P<0.001). The hepatoma cell lines showed strong positive for M-CSF and M-CSF-R production. Both B5 and RE2 displayed a dose-dependent inhibitory effect on the growth and proliferation of hepatoma cells. Conclusion: The study indicates a co-expression model for M-CSF-R in hepatoma cells, suggesting an involvement of M-CSF/M-CSF-R in growth signaling of those malignant cells. The M-CSF/M-CSF-R seems to function through an autonomy mechanism in human hepatoma.

The expression of the products of IGF-Ⅱ,IGF-Ⅱ receptors and CSF-Ⅰ receptors in human primary hepatocellular carcinoma and juxtacancerous liver tissue

The expression of the products of IGF-Ⅱ,IGF-Ⅱ receptors(IGF-Ⅱ-R)and CSF-Ⅰ re-ceptors(CSF-Ⅰ-R)was observed in 17 cases of human primary hepatocellular carcinoma(PHC)and the juxtacancerous liver tissue with immunohistochemistry(ABC),Western blot and North-ern blot technique,It was found that the expression of IGF-Ⅱ,IGF-Ⅱ-R and CSF-Ⅰ-R was signif-icantly higher in PHC than in normal liver tissue and the expression of IGF-Ⅱ and IGF-Ⅱ-R wasremarkably higher in the juxtacancerous liver tissue from PHC patients than in PHC proper.Itwas noteworthy that the expression of IGF-Ⅱ in both the cancer proper and the juxtacancerousliver tissue was characterized by its fetal type.Besides,the expression of CSF-Ⅰ-R was signifi-cantly higher in PHC than in the juxtacancerous liver tissue.It is believed that the abnormal ex-pression of IGF-Ⅱ,IGF-Ⅱ-R and CSF-I-R in PHC and the juxtacaneerous liver tissue might berelated to the autocrine mechanism of human PHC.

Construction of Antisense Transforming Growth Factorβ_1 Gene and Its Effect on the Proliferation by Expression in Osteosarcoma Cells

To construct the antisense transforming growth factorβ1 (TGFβ1) gene and investigate the effect of TGFβ1 autocrine loop blockage on the proliferation of osteosarcoma cells. TGFβ1 cDNA was cloned by RT-PCR from human osteosarcoma cells (MG-63) and inserted into pcDNA3 to construct an antisense expression vector, which was dubbed pcDNA3-TGFβ1(-). MTT was used to detect the proliferation of osteosarcoma cells transfected by antisense TGFβl gene. Our results showed that the proliferation of the transfected osteosarcoma cells was suppressed markedly. It is concluded that TGFβ1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation, which might be helpful for gene therapy of osteosarcoma.

Endothelin increases the proliferation of rat olfactory mucosa cells

The olfactory mucosa holds olfactory sensory neurons directly in contact with an aggressive environment. In order to maintain its integrity, it is one of the few neural zones which are continuously renewed during the whole animal life. Among several factors regulating this renewal, endothelin acts as an anti-apoptotic factor in the rat olfactory epithelium. In the present study, we explored whether endothelin could also act as a proliferative factor. Using primary culture of the olfactory mucosa, we found that an early treatment with endothelin increased its growth. Consistently, a treatment with a mixture of BQ123 and BQ788(endothelin receptor antagonists) decreased the primary culture growth without affecting the cellular death level. We then used combined approaches of calcium imaging, reverse transcriptase-quantitative polymerase chain reaction and protein level measurements to show that endothelin was locally synthetized by the primary culture until it reached confluency. Furthermore, in vivo intranasal instillation of endothelin receptor antagonists led to a decrease of olfactory mucosa cell expressing proliferating cell nuclear antigen(PCNA), a marker of proliferation. Only short-term treatment reduced the PCNA level in the olfactory mucosa cells. When the treatment was prolonged, the PCNA level was not statistically affected but the expression level of endothelin was increased. Overall, our results show that endothelin plays a proliferative role in the olfactory mucosa and that its level is dynamically regulated. This study was approved by the Comité d’éthique en expérimentation animale COMETHEA(COMETHEA C2 EA-45;protocol approval #12-058) on November 28, 2012.

Redefining Human Vitamin D Sufficiency:Back to the Basics

The role of the endocrine vitamin D pathway in regulating the serum calcium concentration in man is well described. In the presence of a low serum calcium level, the vitamin D metabolic pathway is called upon to produce more of the active vitamin D hormone, 1, 25-dihydroxyvitamin D （1, 25D）, via up-regulation of the CYP27b1-hydroxylase activity in the kidney. The consequence is mobilization of skeletal calcium stores to return the circulating calcium level back to the normal range. On the other hand, when the serum calcium level increases, endocrine forces move to suppress renal 1, 25D production and squelch bone resorption. It is now known that vitamin D metabolites also operate in an autocrine, paracrine and intracrine mode to control vitamin D receptor-directed biological responses at the tissue level. Because the metabolism and action of vitamin D occurs at the tissue level in this situation, use of circulating vitamin D metabolites and biomarkers to ascertain the local action of the vitamin D pathway is often misleading. This mini-review seeks to compare and contrast the operation of the vitamin D pathway at the endocrine and tissue level.

THE CELL ORIGIN OF INTERLEUKIN 6 IN THE DISEASE OF MULTIPLE MYELOMA

In order to investigate the cell origin of IL-6 in the disease of multiple myeloma(MM),the IL-6 levels in PBMC culture supernatants from MM and the healthy control were determined with 7TD1 hybridoma growth assay and the ability or MM cell and stroma cell to produce IL-6 was compared.The results showed IL-6 level in PBMC culture supernatants of MM group was evidently higher than that of the control group.Both MM cell and stroma cell could produce IL-6, but IL-6 amount produced by MM cell was 1.6 times as much as that by stroma cell(P<0.01).The results suggest that MM cell is the main cell to produce IL-6.

Crosstalk between glioblastoma and tumor microenvironment drives proneural–mesenchymal transition through ligand-receptor interactions

Glioblastoma(GBM)is the most common intrinsic and aggressive primary brain tumor in adults,with a median survival of approximately 15 months.GBM heterogeneity is considered responsible for the treatment resistance and unfavorable prognosis.Proneural-mesenchymal transition(PMT)represents GBM malignant progression and recurrence,which might be a breakthrough to understand GBM heterogeneity and overcome treatment resistance.PMT is a complicated process influenced by crosstalk between GBM and tumor microenvironment,depending on intricate ligand-receptor interactions.In this review,we summarize the autocrine and paracrine pathways in the GBM microenvironment and related ligand-receptor interactions inducing PMT.We also discuss the current therapies targeting the PMT-related autocrine and paracrine pathways.Together,this review offers a comprehensive understanding of the failure of GBM-targeted therapy and ideas for future tendencies of GBM treatment.

AMF siRNA treatment of keloid through inhibition signaling pathway of RhoA/ROCK1

A keloid(KD)is a benign dermal fibrotic tumor.Treatment of KDs is challenging and the recurrence rate is high;thus,there is an unmet need to explore new target sites and new treatment methods.As a tumor-associated cytokine,autocrine motility factor(AMF)can effectively stimulate the random and directional movement of cells.We first found that AMF was overexpressed in keloid fibroblasts(KFs)and the proliferation and migration of KFs were promoted by AMF stimulation.After treatment with Y-27632,RhoA kinase inhibitor,the proliferation and migration capacity of KFs declined significantly,and type I collagen protein,active RhoA and ROCK1 also were downregulated.In addition,a KD transplantation model was established under the skin of nude mice,with KD intramural injection AMF siRNA,we found that the weight of the KD was smaller than in the control group(P<0.05),KD tissue sections stained by HE and Masson showed that fibers became loose and the blood vessels were visibly reduced.In conclusion,AMF siRNA is expected to be a novel strategy to treat KD by inhibiting signaling pathway of RhoA/ROCK1.

FURTHER STUDY ON CHARACTERIZATION OF 615 MOUSE LEUKEMIC AUTOINHIBITORY ACTIVITY

There have been some reports on autocrine of leukemic cell lines, however, very few on leukemic negative autocrine up to date. It was demonstrated that some of leukemic cell lines could produce Tumor Necrosis Factor(TNF) and also its receptor. We have found an inhibitor from murine leukemia L7811 which could inhibit autologus leukemic cells. Later on leukemic cells from another 615 murine transplantable leukemia L615 was found to have similar autoinhibitor activit. Subsequently, it was confirmed that other

Expression and significance of IL-18 in the bone marrow of patients with hematological diseases

To investigate the levels of IL 18 in the bone marrow of both normal subjects a nd patients with hematological diseases and to determine the possible significan ce of IL 18 in pathogenesis of some hematological malignancies Methods The IL 18 mRNA levels in the bone marrow of 140 patients with hematological dis eases and 15 normal donors were determined by the semi quantitative reverse tra nscriptase polymerase chain reaction (RT PCR) Immunohistochemical method was used to detect IL 18 protein in 12 patients with acute myeloid leukemia (AML) The possible regulation of IL 18 for proliferation of some leukemia cells was investigated using antisense techniques Results IL 18 mRNA levels were obviously higher in the patients with leukemia or other malignant hematological diseases (OMHD) than in normal donors However, no sign ificant difference was found in the level of transcription between patients with iron deficiency anemia (IDA) and normal controls Immunohistochemical method c onfirmed the presence of IL 18 protein in 10 out of 12 AML cases with positive transcription By 3 (4,5 dimethylthiazol 2 yl) 2,5 diphenyl tetrazolium b romide (MTT) assay, IL 18 antisense oligodeoxynucleotides (ASON) clearly inhibi ted the growth of J6 1 and HL 60 cells (42% and 12% inhibited, respectively) i n a dose dependant manner Conclusions IL 18 was detected at elevated levels in the bone marrow of patients with some hematological malignancies, and might be involved in the proliferation of certai n leukemic cells in vivo through an autocrine mechanism

EXPRESSION OF TRANSFORMING GROWTH FACTOR-beta mRNA IN SOME LEUKEMIC CELLS

Bifunctional regulatory effects of TGF-beta for cell proliferation have been focused. TGFbeta becomes a candidate as the autocrine growth inhibitor to some malignant cells. Evidence has been gained in breast cancer cell. However, it has not been proved in leukemia and other tumors. Recently, regulations of external TGF-beta on normal and

ESTABLISHMENT OF A GROUP OF MURINE LEUKEMIC CELL LINES AND INVESTIGATION OF THEIR BIOLOGICAL CHARACTERISTICS

An in vitro-vivo technique for establishment of cell lines on murine leukemia has beendeveloped. Using this method, suppressive T lymphoblastic leukemia L7811-85, L7212-85, non-T, non-B lymphocytic leukemia L1210-86, B lymphocytic leukemia P 388-86 and Friend erythroleu-kemia FLCL cell lines have been established. Incidence of leukemia with these cell lines was 100%. Along with the increase of genera-tions of cell lines, cell growth accelerated, generation time shortened and cloning efficienciesrose. A following up electron microscopic observation on L7811-85 and L7212-85 showed thatthe virus particles were "A" particles in original cells. When they became cell lines in vitro,virus particles increased and transformed into typical "C" particles with budding. An inhibitory activity relevant to leukemic cells on proliferation of leukemic cells hasbeen observed in the supernatant of L7811-85 medium and was regarded as an "autocrine".