Growth factor-receptor pathway interfering treatment by somatostatin analogs and suramin: Preclinical and clinical studies

Interference in growth factor mediated pathways is a new strategy in the treatment of cancer. Somatostatin analogs can inhibit hormone and growth factor secretion, while suramin can block the binding of several growth factors to their receptors. In addition, somatostatin analogs can cause direct growth inhibitory effects after binding to tumoral somatostatin receptors. We tested the efficacy and endocrine effects of chronic treatment with three somatostatin analogs (Sandostatin,^® RC-160 and CGP 15–425) or suramin in several tumor models and in patients with various types of cancer. Treatment with somatostatin analogs caused growth inhibition of breast cancer cells (MCF-7) in vitro, and of rat transplantable pancreatic (50–70% inhibition) and prostatic Dunning tumors (12% inhibition). No tumor growth inhibition was observed with respect to DMBA-induced rat mammary tumors, a transplantable color tumor and a rhabdomyosarcoma in rats. In 34 patients with metastatic pancreatic or gastrointestinal adenocarcinomas chronic Sandostatin treatment caused stable disease in 27% of the patients, but no objective remissions. Somatostatin receptors were found in the responding MCF-7 mammary tumor cells, rat pancreatic tumors and in 20–45% of human breast cancer specimens [J. Steroid Biochem. Molec. Biol. 37 (1990) 1073–1077], but not in rat DMBA-mammary tumors or in 10 human pancreatic adenocarcinomas. Suramin caused significant dose-dependent growth inhibition of human breast cancer cells in vitro and of rat pancreatic tumors in vivo in the presence of plasma levels up to 150 μg/ml. In a preliminary clinical study concerning 11 patients with various tumor types we observed significant hematological, biochemical, endocrine and clinical side effects, but no objective remissions in spite of relevant peak plasma suramin concentrations of 270–330 μg/ml. In conclusion: somatostatin analogs and suramin can cause growth inhibition of various experimental tumors in vitro and in vivo, but the clinical values has to be established for several types of cancer, especially with respect to suramin and suramin-like compounds.     

Emerging principles for the development of resistance to antihormonal therapy: implications for the clinical utility of fulvestrant

We seek to evaluate the clinical consequences of resistance to antihormonal therapy by studying analogous animal xenograft models. Two approaches were taken: (1) MCF-7 tumors were serially transplanted into selective estrogen receptor modulator (SERM)-treated immunocompromised mice to mimic 5 years of SERM treatment. The studies in vivo were designed to replicate the development of acquired resistance to SERMs over years of clinical exposure. (2) MCF-7 cells were cultured long-term under SERM-treated or estrogen withdrawn conditions (to mimic aromatase inhibitors), and then injected into mice to generate endocrine-resistant xenografts. These tumor models have allowed us to define Phase I and Phase II antihormonal resistance according to their responses to E₂ and fulvestrant. Phase I SERM-resistant tumors were growth stimulated in response to estradiol (E₂), but paradoxically, Phase II SERM and estrogen withdrawn-resistant tumors were growth inhibited by E₂. Fulvestrant did not support growth of Phases I and II SERM-resistant tumors, but did allow growth of Phase II estrogen withdrawn-resistant tumors. Importantly, fulvestrant plus E₂ in Phase II antihormone-resistant tumors reversed the E₂-induced inhibition and instead resulted in growth stimulation. These data have important clinical implications. Based on these and prior laboratory findings, we propose a clinical strategy for optimal third-line therapy: patients who have responded to and then failed at least two antihormonal treatments may respond favorably to short-term low-dose estrogen due to E₂-induced apoptosis, followed by treatment with fulvestrant plus an aromatase inhibitor to maintain low tumor burden and avoid a negative interaction between physiologic E₂ and fulvestrant.     

Extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by a benign testicular leydig cell tumor

We present an unusual case with bilateral testicular Leydig cell tumors displaying extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase. Histological examination of a 38-yr-old man infertile due to azoospermia showed him to have bilateral testicular Leydig cell tumors. The in vitro steroidogenic potential of the tumors and their adjacent testicular tissue was evaluated using organ culture. Tumor tissue was found to secrete deoxycorticosterone (DOC), corticosterone (B) and cortisol, which are not produced in normal adult testis, into the medium, while testicular tissue adjacent to the tumors secreted a small amount of DOC and B. Northern blot analysis with cytochrome P-450_C21 complementary DNA (cDNA) and P-450_11β cDNA as probes revealed that the tumor contained a considerable amount of mRNA for P-450_C21 and P-450_11β, while the mRNAs were not detected in the testicular tissues adjacent to the tumors. It is suggested that the high local levels of estrogen and/or progesterone within the Leydig cell tumors and their adjacent testicular tissues induced extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by the tumors and their adjacent testicular tissues.     

TGF-β in the central nervous system: Potential roles in ischemic injury and neurodegenerative diseases

The Transforming Growth Factor-βs (TGF-β) are a group of multifunctional proteins whose cellular sites of production and action are widely distributed throughout the body, including the central nervous system (CNS). Within the CNS, various isoforms of TGF-β are produced by both glial and neural cells. When evaluated in either cell culture or in vivo models, the various isoforms of TGF-β have been shown to have potent effects on the proliferation, function, or survival of both neurons and all three glial cell types, astrocytes, microglia and oligodendrocytes. TGF-β has also been shown to play a role in several forms of acute CNS pathology including ischemia, excitotoxicity and several forms of neurodegenerative diseases including multiple sclerosis, Parkinson's disease, AIDS dementia and Alzheimer's disease.     

Pathology and genetics of adipocytic tumors

Adipocytic tumors are common mesenchymal neoplasms with considerable morphologic and genetic heterogeneity. The fruitful integration of morphology and cytogenetics in the past 15 years has not only enhanced the diagnostic accuracy, but also refined the various pathological classifications and subtypes in these tumors. The current WHO classification includes eleven benign subtypes, one intermediate and five categories of malignant fatty neoplasms with incorporation of relevant genetic findings. Of the benign tumors, lipomas have been extensively analyzed by chromosome banding which has shown that their cytogenetic patterns are heterogeneous. Still aberrations involving 12q13-->q15, 6p23-->p21 and loss of material from 13q are common and consistent findings. Among the malignant tumors, the t(12;16)(q13;p11) resulting in the fusion of DDIT3 and FUS genes is the hallmark of myxoid and round cell liposarcoma and is used as a highly specific and sensitive marker of this entity. The tumor in the intermediate group, atypical lipomatous neoplasm/well-differentiated liposarcoma which poses morphologic challenges due to close histological similarity to benign lipomas shows characteristic supernumerary rings and giant rod chromosomes due to amplification of the 12q14-->q15 region often involving the MDM2 oncogene. This review will focus on the pathological features of the various adipocytic tumors and relevant genetic findings reported in the literature.     

The diversity of p53 mutations among human brain tumors and their functional consequences

The p53 tumor suppressor is implicated in cell cycle control, DNA repair, replicative senescence and programmed cell death. Inactivation of the p53 contributes to the wide range of human tumors, including glial neoplasms. In this review, we describe the regulation and biochemical properties of p53 protein that may explain its ability to activate various genetic programs underlying cellular responses to stress conditions. The overall spectrum of p53 mutations is rather shared between tumor types indicating that these mutations are not tumor type-specific. However, there is one example of germ-line mutation of p53 gene (the deletion of the codon 236) that is associated with a familiar brain tumor syndrome. We compare the frequency and type of most common mutations among various brain tumours (focusing on glioblastomas) and their consequences on protein functions. Furthermore, we discuss the most promising approaches of potential brain tumor therapy, including an adenovirus-mediated p53 gene transfer. Human glioblastomas are highly sensitive to the effects of p53 activity when the wild-type p53 is introduced ectopically. It suggests that the genetic or pharmacological modulation of the p53 pathway is potentially important strategy in the treatment of human cancers.     

Progesterone antagonists: Tumor-inhibiting potential and mechanism of action

A new approach for the treatment of breast cancer could be the use of progesterone antagonists. These compounds were originally developed for the inhibition of progesterone-dependent processes and have been shown to be effective in inhibition of nidation and interruption of pregnancy. Although the roles of progesterone and the progesterone receptor in control of cell growth remain unclear, it was found in progesterone receptor positive mammary carcinoma cell lines that the antiprogestin, Mifepristone, had an inhibitory effect on cell growth-and a growth-inhibiting action on the DMBA-induced mammary carcinoma of the rat. We have shown that the progesterone antagonists, Onapristone and ZK 112993, which possess a reduced antiglucocorticoid activity compared to Mifepristone, exert a strong tumor-inhibiting effect in a panel of hormone-dependent mammary tumor models. The effects of these compounds were in some systems superior to those of tamoxifen or high dose progestins and comparable to ovariectomy. Although prerequisites for their antiproliferative potency are an affinity to the progesterone receptor as well as a sufficient number of available receptors in the tumors, the strong tumor inhibiting potential of the antiprogestins cannot be explained by a classical antihormonal mechanism. Surprisingly, the antitumor activity is evident in spite of elevated serum levels of ovarian and pituitary hormones. It was established by morphometric procedures that treatment with Onapristone triggers differentiation of the mitotically active polygonal tumor epithelial cell towards secretory active glandular structures and acini. All our quantitative light and electron microscopic data indicate that the antitumor action of antiprogestins is accompanied by the initiation of terminal differentiation leading to (apoptotic) cell death. Finally, our flow cytometry studies revealed an accumulation of the tumor cells in the G₀G₁ phase of the cell cycle, which may result from induction of differentiation since a differentiation-specific G₁ arrest has already been proposed for other stem cell systems. It can be concluded from these data that the progesterone receptor antagonists differ in their mode of action from compounds used in established endocrine treatment strategies for mammary carcinoma. The ability of progesterone antagonists like Onapristone to reduce the number of cells in S-phase may offer a significant clinical advantage, since it is established that the S-phase fraction is a highly significant predictor of disease-free survival among axillary node-negative patients with diploid mammary tumors.     

P-Akt expression distinguishes two types of malignant rhabdoid tumors

Highly aggressive pediatric malignant rhabdoid tumors (MRT) arise in the kidney and central nervous system (CNS) with no curative treatment available. Multiple studies have shown that inactivation of the SNF5 tumor suppressor gene occurs in virtually all MRTs. However, few studies have addressed whether additional genetic events may contribute to MRT development. In this report, we demonstrate that phosphorylated Akt (P-Akt) is expressed in a subpopulation of cells in at least 10% of primary rhabdoid tumors as well as at high levels in three MRT cell lines. Similar to other high P-Akt expressing tumor cell lines, MRTs have decreased sensitivity to p21 induced growth arrest. Therefore, P-Akt expression may distinguish between two types of MRTs. Because drugs directed against the PI3-K/Akt have shown promise in clinical trials for other tumor types, they may prove useful for treatment of patients with P-Akt positive MRTs. P-Akt expression also provides a potential mechanistic link between these pediatric tumors and adult malignancies.     

Effects of progestagens and Org OD14 in in vitro and in vivo tumor models

Sex steroids, in particular estradiol (E2) and progesterone (P4), play, together with other hormones and growth factors, a role in the development of normal breast tissue. The effect of four progestagens (norethisterone, 3-ketodesogestrel, gestodene and P4) and Org OD14, a steroid with weak estrogenic, progestagenic and androgenic properties were studied on growth of breast tumor cells in vitro using two subclones of MCF-7 (H and A) and T47D (S and A) cells. In addition, we investigated the effects of 3-ketodesogestrel, gestodene and Org OD14 on the growth of 7,12-dimethyl-benz(a)anthracene(DMBA)-induced mammary tumors in rats. In the in vitro assays with MCF-7 cells norethisterone, 3-ketodesogestrel and gestodene stimulated growth only at high doses (10⁻⁷ M), whereas P4 had no effect. Gestodene was more potent than 3-ketodesogestrel and norethisterone. Org OD14, stimulated cell growth at a dose of 10⁻⁸ M, while E2 is active at 10⁻¹⁰ M. In T47D-A cells similar effects were found, but the subclone S did not respond to the progestagens and Org OD14. The two T47D subclones also reacted differently to progestagens during growth stimulation with E2. In T47D-S the progestagens and Org OD14 inhibited, while in T47D-A these compounds did not modulate the effect of E2. In the DMBA model we found that gestodene and 3-ketodesogestrel were able to inhibit tumor growth to the same extent. Surprisingly, Org OD14 was even more effective in the DMBA model using the therapeutic approach. Using the prophylaxic approach tumor development was delayed and tumor growth was strongly suppressed. The inhibitory effects of Org OD14 on tumor growth in the DMBA model may be attributed to its mixed hormonal profile. From these studies we conclude that different cell lines and even subclones thereof respond quite differently to steroids. Both in vitro and in vivo studies are required to judge whether synthetic steroids might be involved in an increased risk for the development of breast tumors.     

Malignant transformation in human chondrosarcoma cells supported by telomerase activation and tumor suppressor inactivation.

Human chondrosarcomas do not respond to current chemotherapies or radiation therapy, and their size and histological appearance do not reliably predict the risk of local recurrence and metastases, making selection of surgical treatment difficult. Identifying mechanisms responsible for the proliferation and invasive behavior of these tumors would be of immense clinical value. We hypothesized that telomerase expression is one of these mechanisms. We detected telomerase expression in 7 of 16 chondrosarcomas, but cells cultured from telomerase-negative chondrosarcomas acquired strong telomerase activity and lost tumor suppressor activity after their establishment in culture. These changes were associated with accelerated indefinite cell proliferation, morphological transition, and increased invasive activity, indicating that telomerase activation and loss of cell cycle control leads to the emergence of aggressive cells from chondrosarcoma cell populations. These observations may lead to better understanding of the factors responsible for malignant transformation, local recurrence, and metastases of cartilage neoplasms.     

Insight into generation of induced mesenchymal stem cells from induced pluripotent cells

Mesenchymal stem cells(MSCs)have the potential for use in cell-based regenerative therapies.Currently,hundreds of clinical trials are using MSCs for the treatment of various diseases.However,MSCs are low in number in adult tissues;they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures.These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications.As a result,novel methods to generate induced MSCs(iMSCs)from induced pluripotent stem cells have been explored.The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro.In addition,it is important to compare iMSCs with primary MSCs(isolated from adult tissues)in terms of their safety and efficacy.Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.     

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.     

Vitamin D: A modulator of cell proliferation and differentiation

1,25-Dihydroxyvitamin D₃, [1,25(OH)₂D₃], the biologically most active metabolite of vitamin D₃, is involved in the regulation of calcium homeostasis and bone metabolism. Recently, receptors for 1,25(OH)₂D₃ have also been shown in cells and tissues not directly related to calcium homeostasis. Experimental data obtained with leukemic and cancer cell lines, both in vitro and in vivo, showed the effects of 1,25(OH)₂D₃ on cell differentiation and proliferation. However, high doses of the sterol have to be used to observe these effects. Additional studies are needed to establish whether 1,25(OH)₂D₃ or suitable analogues have a therapeutic potential in malignant diseases without unacceptable toxicity like the development of hypercalcemia.     

Current advances of stem cell-based therapy for kidney diseases

Kidney diseases are a prevalent health problem around the world. Multidrug therapy used in the current routine treatment for kidney diseases can only delay disease progression. None of these drugs or treatments can reverse the progression to an end-stage of the disease. Therefore, it is crucial to explore novel therapeutics to improve patients’ quality of life and possibly cure, reverse, or alleviate the kidney disease. Stem cells have promising potentials as a form of regenerative medicine for kidney diseases due to their unlimited replication and their ability to differentiate into kidney cells in vitro. Mounting evidences from the administration of stem cells in an experimental kidney disease model suggested that stem cell-based therapy has therapeutic or renoprotective effects to attenuate kidney damage while improving the function and structure of both glomerular and tubular compartments. This review summarises the current stem cell-based therapeutic approaches to treat kidney diseases, including the various cell sources, animal models or in vitro studies. The challenges of progressing from proof-of-principle in the laboratory to widespread clinical application and the human clinical trial outcomes reported to date are also highlighted. The success of cell-based therapy could widen the scope of regenerative medicine in the future.     

Cancer stem cells and survival pathways

Gliomas and medulloblastomas are the most frequent malignant brain tumors in adult and children respectively. Although both tumors arise in the CNS, there is a significant difference in their therapeutic response, resulting in medulloblastomas as being relatively curable, while glioblastomas are basically incurable. During the last decade several reports have demonstrated the existence of cancer stem cells in brain tumors, their location and their response to treatment. We have recently described the therapeutic response of medulloblastomas to radiation in their native microenvironment, describing how p53 and PI3K signaling pathway leads to nestin-expressing cells in the perivascular stem cell niche evading cell death while the tumor-bulk succumbs to apoptosis 1. It remains to be determined whether this mechanism of tumor resistance applies to the more complex stem-cell niche and tumor bulk of gliomas.     

Preclinical models for the evaluation of targeted therapies of metastatic disease

It has been estimated that approx 60–70% of cancer patients harbor overt or subclinical metastases at diagnosis, and it is the eradication of such systemic disease that largely determines survival. Preclinical tumor model systems employed to evaluate potential new treatment strategies should aim to represent the process and patterns of metastasis of their clinical counterparts as closely as possible. Severe combined immune-deficient (SCID) andnu/nu mice have been extensively used as hosts for the growth of human tumor cell lines and in some cases fresh tumor material. However, in most instances the resulting neoplasms fail to metastasize, and the aberrant immune systems of such animals has limited their use mainly to passive therapies of localized disease. Recently, the development of specially selected tumor variants and the use of appropriate orthotopic sites for implantation has provided several models in which dissemination can be demonstrated. Where the gene coding for a potential target antigen has been cloned, and where its overexpression or mutation is associated with malignancy (e.g., c-erbB-2, H-ras), transgenic mice may yield tumors that will develop in these immunocompetent hosts. In some cases such tumors exhibit metastasis. A third approach is to transfect human genes of interest into appropriate rodent tumors expressing the desired metastatic phenotype. These various approaches are compared with particular reference to mammary carcinoma biology.     

Survival and neurotransmitter plasticity in cultured rat colonic myenteric neurons

The enteric nervous system is of great importance for maintenance and proper function of the gastrointestinal tract. The aim of this study was to quantify myenteric neuronal subpopulations expressing calcitonin gene-related peptide (CGRP), galanin, neuropeptide Y (NPY), somatostatin, vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) in rat colon in vivo and after culturing. Further we investigated if culturing in the presence of CGRP, galanin, VIP, S-nitroso-N-acetyl-d,l-penicillamine (SNAP, a NO donor) or N-nitro-l-arginine methyl ester (l-NAME, a NOS inhibitor) affect neuronal survival.

After 4 days of culturing the proportions of neurons expressing CGRP, NPY, somatostatin or VIP increased as compared to in vivo, while the proportions of neurons expressing galanin or NOS did not change. Neuronal survival was unaffected after culturing in media enriched with CGRP, galanin, VIP, SNAP or l-NAME. Neither did addition of CGRP, galanin nor VIP to the cultures affect the relative numbers of neurons expressing CGRP, galanin or VIP respectively. Addition of SNAP or l-NAME did not change the percentage of neurons expressing NOS.

In conclusion, cultured rat colonic myenteric neurons increase their expression of CGRP, NPY, somatostatin and VIP, suggesting that these neuropeptides are of importance for neuronal survival.     

In vivo manipulation of human breast cancer growth by estrogens and growth hormone: Kinetic and clinical results

Since 1983, a series of experimental and clinical studies have been carried out on the possibility of enhancing the chemotherapy effectiveness in breast cancer by expanding the fraction of cycling cells. Theoretically estrogens should recruit breast cancer cells and this fact should result in a higher killing efficiency of antiproliferative drugs. Actually it has been clearly shown, by means of the thymidine labeling index and primer-dependent -DNA polymerase assay, that low doses of diethylstilbesterol are able to increase the tumor proliferative activity of human breast cancer in vivo (estrogenic recruitment).

Three randomized trials have been carried out (one in locally advanced and two in metastatic breast cancer) comparing conventional polichemotherapy vs chemotherapy with estrogenic recruitment. Only limited advantages have been observed in these trials.

Searching for new modalities of kinetic manipulation of tumors, recombinant human growth hormone has been employed in a pilot study: the preliminary results indicate that it largely enhances tumor proliferative activity, suggesting the possibility of employing a growth factor system to increase chemosensitivity.     

Immunohistochemical detection of nestin in pediatric brain tumors.

Nestin is an intermediate filament protein (IFP) expressed in undifferentiated cells during CNS development and in CNS tumors. Previous studies have arrived at different conclusions in terms of which types of CNS tumors express nestin. In this report we establish an immunohistochemical protocol using antigen retrieval, which significantly enhances staining with two polyclonal anti-nestin antisera, #130 and #4350. The staining pattern was identical for the two nestin antisera and very similar to that of vimentin, while glial fibrillary acidic protein (GFAP), immunoreactivity was absent from 9.5-week-old forebrain. The current study of 20 primary CNS tumors from pediatric patients included seven ependymomas, seven primitive neuroectodermal tumors (PNETs), five pilocytic astrocytomas, and one glioblastoma multiforme (GBM). All these tumors expressed nestin to various extents, in contrast to five brain metastases tested. Strong nestin immunoreactivity was found in malignant primary CNS tumors, whereas benign pilocytic astrocytomas showed low but consistent nestin expression. In all tumors nestin immunoreactivity was confined to the cytoplasm of tumor cells and was co-expressed with astrocyte markers vimentin, GFAP, and S-100. Vascular endothelial cells of all neoplasms also showed marked immunoreactivity for nestin and vimentin, whereas they were negative for GFAP and S-100. In conclusion, antiserum #4350 detected nestin in formalin-fixed, paraffin-embedded tissue sections by heat-induced antigen retrieval immunohistochemistry. Nestin was expressed in both highly malignant and low malignant gliomas, indicating the potential use of nestin as a diagnostic tumor marker in surgical pathology.