N-Methyl-(2S,4R)-trans-4-hydroxy-L-proline isolated Sideroxylon obtusifolium attenuates TPA-induced irritant contact dermatitis in mice.

Dermatitis is defined as a set of inflammatory diseases that affect the skin, with varied causes. Among the different types of dermatitis, contact dermatitis is the most prevalent. Although the current therapy is often effective, it is associated with adverse effects and the possibility of drug tolerance. N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline is a L-proline amino acid derivative found in the leaves of Sideroxylon obtusifolium, a species traditionally used to treat inflammatory diseases. The aim of this study was to investigate the topical anti-inflammatory effect of N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline (NMP) in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced irritant contact dermatitis in mice. Topically administered NMP, at doses of 0.03 - 0.50 mg/ear, reduced TPA-induced ear edema and neutrophil migration, as evidenced by low tissue myeloperoxidase activity and verified by histological examination. In addition, NMP (0.06 mg/ear) reduced tissue levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, INF-γ and MCP-1) and of the anti-inflammatory cytokine IL-10, and reduced gene expression of TNF-α, IL-6 and IL-1ß increased by TPA. The data suggest that N-methyl-(2S, 4R)-trans-4-hydroxy-L-proline acts as a topical anti-inflammatory agent that decreases the expression of inflammatory cytokines, making it useful for the treatment of skin inflammation. Further investigations are necessary for its development as a therapeutic agent.

Anti-proliferative and anti-inflammatory effects of the application of baclofen cream, a GABAB receptor agonist, on skin inflammation in mice.

Previous studies have demonstrated the role of γ-aminobutyric acid type B (GABAB) receptors in skin-related conditions and pain. However, most studies have focused on the main effects of GABAB on the central nervous system. Therefore, this study has aimed to determine the potential topical anti-inflammatory and anti-proliferative effects of baclofen cream in an inflammatory skin disease model. The effects of the baclofen cream were evaluated using acute and chronic models of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mouse ears. Histological and immunohistochemical evaluations were performed using an ear oedema assay. The effect of baclofen on keratinocyte proliferation was assessed in PAM212, the murine keratinocyte cell line. The results demonstrate that a single topical application of 5% baclofen, 7.5% baclofen, and 1% dexamethasone each inhibited acute TPA-induced ear oedema (58.94 ± 6.14%, 47.73 ± 11.26%, and 87.33 ± 4.59%, respectively). These results were confirmed by histological analysis. In the chronic model, baclofen (5%) and dexamethasone (1%) each inhibited ear oedema and the maximum inhibitory effect was reached at the end of the experiment (9th day of TPA application) with a percentage inhibition of 54.60 ± 6.15% for baclofen and 71.68 ± 3.45% for dexamethasone, when compared to the vehicle. These results were confirmed by histological analysis. Baclofen and dexamethasone also reduced proliferating cell nuclear antigen expression by 62.01 ± 6.65% and 70.42 ± 6.11%, respectively. However, baclofen did not inhibit keratinocyte proliferation in PAM212 cells. In conclusion, these results demonstrate that baclofen exhibits notable topical antiproliferative and anti-inflammatory properties and could be a potential therapeutic alternative for treating inflammatory and proliferative skin diseases.

Activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway overcomes cisplatin resistance in ovarian carcinoma cells.

OBJECTIVE: This study was aimed to elucidate the roles of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3K)/Akt pathways in regulating cytotoxicity induced by cisplatin (CDDP) in ovarian carcinoma cells. METHODS: We treated 7 ovarian cancer cell lines with CDDP alone or with CDDP and either a PI3K inhibitor (LY294002), a MEK inhibitor (PD98059), or a MEK/ERK activator (phorbol 12-myristate 13-acetate [PMA]) and assessed cell viability, expression of MEK/ERK and PI3K/Akt, cell cycle distribution, and apoptosis. We also investigated the effect of combination treatment on survival in a xenograft model. RESULTS: The cell lines showed half-maximal inhibitory concentrations (IC50) of CDDP from 2.4 to 26.9 µmol/L. KFr, a CDDP-resistant cell line developed from KF cells, showed an IC50 of CDDP of 9.6 µmol/L. Five of the cell lines with IC50 values of 9.6 µmol/L or greater were defined as CDDP-resistant. Cisplatin and LY294002 had an additive effect on inhibiting cell growth, and CDDP and PD98059 had and antagonistic effect on cell growth in all cell lines. In CDDP-resistant cells, CDDP and PMA dramatically suppressed the cell growth, up-regulated the expression of phosphorylated ERK and cleaved caspase-9, down-regulated the expression of checkpoint kinases, and increased the proportion of cells in the synthesis-phase fraction and apoptotic cells. The treatment of nude mice with CDDP and PMA prolonged survival in an ovarian cancer xenograft model. CONCLUSIONS: The present study indicates that further study is warranted to determine the effectiveness of combination treatment with CDDP and PMA for platinum-resistant ovarian carcinoma.

The role of exosomal miR-181b in the crosstalk between NSCLC cells and tumor-associated macrophages.

BACKGROUND: It has been reported that tumor-associated macrophages (TAMs) participate in modulating the progression of cancer in the tumor microenvironment. However, the crosstalk between TAMs and non-small cell lung cancer (NSCLC) is still unclear. OBJECTIVE: We investigated whether NSCLC-derived exosomes could affect TAMs, which feedback modulated progression of NSCLC. METHODS: MiR-181b expression was measured by RT-PCR. Human THP-1 monocyte was differentiated into macrophages with phorbol myristate acetate, which were further identified by transmission electron microscopy and western blot. Macrophage M1 and M2 polarizations were detected by flow cytometry, RT-PCR and western blot. Proliferation, migration, and invasion of NSCLC cells treated with conditioned mediums were detected by EdU and Transwell assays. RESULTS: We demonstrated that miR-181b was up-regulated in exosomes derived from NSCLC patients' serum and NSCLC cells. MiR-181b could be transferred to macrophages via exosomes in the co-culture system of macrophages and NSCLC cells, which promoted macrophage M2 polarization. Further examinations revealed that exosomes derived from NSCLC cells could enhanced macrophage M2 polarizations by regulating miR-181b/JAK2/STAT3 axis, and silencing miR-181b in NSCLC cells and JAK2 inhibitor used in macrophages could reverse the effects. Importantly, the conditioned medium of macrophages treated with NSCLC cell-derived exosomes could promote NSCLC cell proliferation, migration, and invasion. Silencing miR-181b in NSCLC cells and JAK2 inhibitor used in macrophages could block the effects. CONCLUSIONS: All of these results indicated that exosomal miR-181b participated in the crosstalk between NSCLC cells and TAMs, providing potential therapeutic targets for NSCLC.

Inhibitory effects of 7,7'-bromo-curcumin on 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation.

This study aimed to determine the capacity of 7,7'-bromo-curcumin (CUR-Br), a curcumin analogue with higher chemical stability than curcumin (CUR), in the suppression of mouse ear edema. Male CD-1 mice were topically pre-treated with either CUR or CUR-Br for 30 min prior to an application of 12-O-tetradecanoylphorbol-13-acetate. After 6 h, mice were killed, and ear punches were measured for their weight and thickness as a marker of edema and inflammation. CUR-Br demonstrated a higher anti-inflammatory efficacy compared to CUR. CUR and CUR-Br at 1.0 µmol suppressed the TPA-induced increase in the ear weight by 26.0% and 57.2%, and decreased TPA-induced increase in the ear thickness by 22.2% and 84.7%, respectively. The inhibitory effects of Cur-Br were associated with decreased levels of inflammatory cytokines (IL-1ß, IL-2, IL-6, KC/GRO, IL-10, IL-17, and IL-23). In addition, CUR-Br significantly downregulated expression of pro-inflammatory signaling proteins such as p-STAT3, STAT3, PI3K, AKT, p-p65, and COX-2. Overall, our results demonstrated that the curcumin analogue, CUR-Br, showed stronger anti-inflammatory properties than CUR in inhibiting TPA-induced inflammatory response in mouse skin.

Chemotherapy agents induce tartrate-resistant acid phosphatase 5a contributing to the symptom distress in lung cancer patients.

Tartrate-resistant acid phosphatase 5a (TRACP5a) is mainly secreted by activated macrophages in chronic inflammation. Serum TRACP5a is associated with symptom distress in lung cancer patients during chemotherapy. Therefore, this study aimed to investigate whether chemotherapy drugs modulate TRACP5a as an inducible marker for symptom distress in lung cancer patients during chemotherapy. In clinical analysis, lung cancer participants completely received the six-cycle chemotherapy process (n = 42). Clinical determinations for TRACP5a, C-reactive protein (CRP), interleukin-6 (IL-6), white blood cells, monocytes, and hemoglobin were analyzed at six time points: BL, C1d8, C2d1, C4d1, C4d8, and Ed28. Meanwhile, five questionnaires for fatigue, sleep disturbance, pain, depression, and confusion were finished before drug treatment. For monocyte-to-macrophage differentiation, THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA). TRACP5a secretion in THP-1 cells was determined at the following days up to 6 days after 1-day incubation of chemotherapy drugs by dot blotting. Clinical analysis revealed that TRACP5a significantly increased at C1d8 and C4d8, but dropped at C2d1 and Ed28. CRP and IL-6 displayed a broad-range variation, resulting in no significant difference among the assessment time points. In contrast, monocytes decreased at C1d8 and C4d8, but rose again at C2d1 and Ed28. In symptom distress, the changes only in fatigue and sleep disturbance were positively associated with the trend in TRACP5a. In PMA-treated THP-1 cells, TRACP5a significantly increased after stimulation with gemcitabine and paclitaxel. Taken together, induction of TRACP5a by chemotherapy drugs might be generated from monocyte-differentiated macrophages, further causing clinical symptom distress in lung cancer patients.

Autophagy and autophagic cell death are next targets for elimination of the resistance to tyrosine kinase inhibitors.

Autophagy, a cellular degradation system has been demonstrated in some hematopoietic malignant cell lines, but there is much still remaining to be known about its role and the mechanisms. We observed the excessive autophagy in chronic myelogenous leukemia (CML) cell line, K562, associated with treatment of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which can induce K562 cells to differentiate into megakaryocytic lineage. Confocal microscopic analysis demonstrated that autophagic cells did not express a megakaryocyte marker, the CD41 molecule, indicating that the autophagy was independent of megakaryocytic differentiation. After remarkable autophagic degradation, the cells finally underwent autophagic cell death (APCD). On the other hand, a block of TPA-induced autophagy by chloroquine rapidly promoted cell death that was not APCD. This result suggested that autophagy regulated two mechanisms in K562 cells: both the cell survival system and APCD. To confirm that autophagy regulates the cell survival system in K562 cells, imatinib was used to induce cell death in K562 cells. Autophagy has not been considered during imatinib treatment; nonetheless, co-treatment with imatinib and chloroquine markedly enhanced imatinib-induced cell death, compared to K562 cells treated only with imatinib. Furthermore, imatinib-resistant cell lines, BaF3/T315I and BaF3/E255K, also underwent cell death by co-treatment with imatinib and chloroquine. From these data, we concluded that autophagy is deeply related to the cell survival system and that inhibition of autophagy accelerates TPA- or imatinib-induced cell death. The block of autophagy could be a new strategy in the treatment of CML.

A phase I clinical trial of 12- O-tetradecanoylphorbol-13-acetate for patients with relapsed/refractory malignancies.

Phorbol esters activate protein kinase C and modulate a variety of downstream cell signaling pathways. 12-O-tetradecanoylphorbol-13-acetate (TPA) is a phorbol ester that induces differentiation or apoptosis in a variety of cell lines at low concentrations. A phase I dose escalation trial of TPA was undertaken for patients with relapsed or refractory malignancies. The starting dose was 0.063 mg/m2 and most patients were treated with an intravenous infusion of TPA on days 1-5 and 8-12 followed by a 2-week rest period prior to retreatment. Thirty-five patients were treated. A biological assay was used to monitor levels of TPA-like activity in the blood after treatment. Serious adverse events included individual episodes of gross hematuria, a grand mal seizure, syncope, and hypotension. Many patients had transient fatigue, mild dyspnea, fever, rigors, and muscular aches shortly after the infusion. Dose-limiting toxicities included syncope and hypotension at a dose of 0.188 mg/m2. Only a single patient had evidence of tumor response. These studies establish 0.125 mg/m2 as the maximally tolerated dose when TPA is administered on this schedule.

Inhibitory effect of 12-O-tetradecanoylphorbol-13-acetate alone or in combination with all-trans-retinoic acid on the growth of LNCaP prostate tumors in immunodeficient mice.

Clinically achievable concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA; 0.16-0.32 nM) and all-trans-retinoic acid (ATRA; 0.5-1 micro M) had a synergistic inhibitory effect on the growth of cultured LNCaP prostate cancer cells, and apoptosis was markedly stimulated. In additional studies, NCr immunodeficient mice received s.c. injection with LNCaP cells in Matrigel. After 4-6 weeks, mice with well-established tumors received i.p. injection with vehicle, TPA (0.16 nmol/g body weight), ATRA (0.5 nmol/g body weight), or TPA+ATRA in vehicle once a day for 46 days. Tumor growth occurred in all of the vehicle-treated control mice. The percentage of animals with some tumor regression after 21 days of treatment was 0% for the control group, 31% for the ATRA group, 62% for the TPA group, and 100% for the TPA+ATRA group (13 mice/group). Although treatment of the mice with TPA or TPA+ATRA continued to inhibit tumor growth for the duration of the 46-day study, treatment of the mice with ATRA alone did not inhibit tumor growth beyond 28 days of daily injections (6 mice/group). Mechanistic studies indicated that treatment of the mice with TPA or TPA+ATRA for 46 days increased apoptosis in the tumors, and treatment with TPA+ATRA also decreased the mitotic index. Because the dose of TPA used in this study was effective and resulted in clinically achievable blood levels, clinical trials with TPA alone or in combination with ATRA in patients with prostate cancer may be warranted.

Successful treatment of murine melanoma with bryostatin 1.

Bryostatins are a novel class of protein kinase C activators which were isolated from the marine bryozoan Bugula neritina and found to possess both antineoplastic and immunoenhancing properties. In this report, we examined the relationship between the in vivo and in vitro antineoplastic effects of bryostatin 1. The in vivo antitumor activity of bryostatin 1 was demonstrated in a B16 melanoma pulmonary metastases model, in which treatment of tumor-bearing C57BL/6 mice with 5 days of bryostatin 1 resulted in a significant reduction in of the number of lung nodules (control, 87; bryostatin, 7). There was a clear dose-response effect, with the optimal antimelanoma dose being 100 micrograms/kg/day, but even low doses of bryostatin 1 of 1 micrograms/kg/day resulted in a 53% reduction in the number of metastases. Although bryostatin 1 shares many biological properties with the phorbol esters, parallel treatment with 12-O-tetradecanoyl 13-phorbol acetate was ineffective against B16 melanoma in vivo. Using a clonogenic assay, bryostatin 1 was found to have a direct antiproliferative effect against B16 melanoma. This inhibition occurred at relatively high bryostatin 1 concentrations (10(-6) M), in comparison with a sensitive cell line REH (10(-10) M). Treatment of mice with bryostatin 1 or preincubation of normal spleen cells with bryostatin 1 failed to enhance nonspecific cell-mediated cytotoxicity against B16 melanoma in vitro. Moreover, bryostatin 1 was found to inhibit both natural killer cell activity and interleukin 2 generation of lymphokine-activated killer cells. Thus, a role for an in vivo immune enhancement mechanism as the basis for the antimelanoma activity observed with bryostatin 1 cannot be invoked from these experiments. These findings indicate that bryostatin 1 may act directly on the B16 melanoma pulmonary metastases. The precise mechanism whereby bryostatin exerts its antimelanoma effects remains unclear.

Differential effects of phorbol esters on normal myeloid precursors and leukemic cells: basis for autologous bone marrow reconstitution in acute nonlymphocytic leukemia using phorbol ester-treated bone marrow from patients in remission.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces macrophage-like differentiation of HL60 cells and cells from patients with acute nonlymphocytic leukemia (ANLL). We assessed the use of TPA as a means of eradicating residual leukemia from remission bone marrow prior to autologous bone marrow reconstitution. A 30-min incubation with TPA led to marked growth arrest in HL60 cells and in cells from most patients with acute myelogenous leukemia and acute myelomonocytic leukemia, whereas cells from most patients with acute promyelocytic leukemia and acute undifferentiated leukemia demonstrated a lesser degree of growth arrest. Freezing and thawing, a necessary step in autologous reconstitution, had no effect on the cessation of proliferation induced in HL60 or ANLL cells preincubated with TPA for 30 min. Virtually normal myeloid precursor growth occurred in normal or remission bone marrow cells preincubated with TPA and then frozen and thawed. Based on these observations, two patients with advanced ANLL in remission underwent marrow ablative therapy followed by autologous reconstitution using TPA-treated bone marrow. Limited normal hematopoiesis was reestablished in both patients, although they subsequently experienced leukemic relapse. These studies demonstrate that in ANLL cells, TPA stimulates growth arrest; in contrast, hematopoiesis is able to proceed both in vitro and in vivo.

Mitogen-activated protein kinase pathway is dispensable for microtubule-active drug-induced Raf-1/Bcl-2 phosphorylation and apoptosis in leukemia cells.

Raf-1 activation and Bcl-2 hyperphosphorylation following treatment with paclitaxel (Taxol) or other microtubule-active drugs is associated with mitotic arrest. Here we show that microtubule-active drugs do not activate the mitogen-activated protein kinase (MAPK) pathway in leukemia cells. PD98059, a MEK inhibitor, and SB202190, a p38 MAP kinase inhibitor, do not abrogate Bcl-2 phosphorylation nor apoptosis. Simultaneously with PARP cleavage, paclitaxel induces cleavage of Bcl-2 protein yielding a potentially pro-apoptotic 22 kDa product. In comparison, the stimulation of Raf-1 by phorbol ester (TPA) activates the MAPK pathway, causes MAPK-dependent p21WAF1/CIP1 induction, Rb dephosphorylation and growth arrest without Bcl-2 phosphorylation or apoptosis. Like TPA, cAMP induces p21WAF1/CIP1 but does not cause Bcl-2 phosphorylation. MEKK1 and Ras, upstream activators of JNK and ERK MAPK, also fail to induce Bcl-2 hyperphosphorylation. Although Lck tyrosine kinase has been recently implicated in Raf-1 activation during mitotic arrest, microtubule-active drugs induce Raf-1/Bcl-2 hyperphosphorylation and apoptosis in a Lck-deficient Jurkat cells. Therefore, microtubule-active drugs induce apoptosis which is associated with Raf-1 and Bcl-2 phosphorylation and Bcl-2 cleavage but is independent of the MAPK pathway. In contrast, TPA-activated MAPK pathway causes p21WAF1/CIP1-dependent growth arrest without apoptosis.

Administration of a phorbol ester to patients with hematological malignancies: preliminary results from a phase I clinical trial of 12-O-tetradecanoylphorbol-13-acetate.

PURPOSE: Phorbol esters are capable of inducing a broad range of cellular effects,including the maturation/differentiation of hematopoietic cell lines (E. Huberman and M. F. Callaham, Proc. Natl. Acad. Sci. USA, 76: 1293-1297, 1979; J. Lotem and L. Sachs, Proc. Natl. Acad. Sci. USA, 76: 5158-5162, 1979; G. Rovera et al., Proc. Natl. Acad. Sci. USA, 76: 2779-2783, 1979; H. P. Koeffler, J. Clin. Investig., 66: 1101-1108, 1980). The ability to induce this differentiation at very low concentrations stimulated investigators to administer a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), to patients with myeloid leukemias in the People's Republic of China (Z. T. Han et al., Proc. Natl. Acad. Sci. USA, 95: 5357-5361, 1998). The tolerability of this therapy in China prompted Phase I studies of TPA in the United States. The purpose of this report is to demonstrate the tolerance of TPA at doses that result in detectable biological activity in blood and malignant cells. EXPERIMENTAL DESIGN: TPA was administered to patients with relapsed/refractory hematological malignancies. RESULTS: Phenotypic effects were detected in malignant cells and TPA-associated biological activity was present in blood for up to several hours after the infusion. CONCLUSIONS: These studies confirm the feasibility of TPA administration to humans and establish the foundation for the development of phorbol esters as therapy for patients with a variety of malignant and nonmalignant disorders.

Pharmacological preconditioning in rabbit myocardium is blocked by chloride channel inhibition.

OBJECTIVES: We have recently proposed that chloride (Cl(-)) channels contribute to ischemic preconditioning (IPC) in the myocardium. To further evaluate this hypothesis, we investigated the role of Cl(-) channels in pharmacological preconditioning. METHODS: Isolated rabbit cardiomyocytes and isolated buffer-perfused rabbit hearts were initially preconditioned with a 10 min exposure to either an adenosine receptor agonist [2-chloro-N(6)-cyclopentyladenosine (CCPA, 200 nM) and/or N(6)-2-(4-aminophenyl)ethyladenosine (APNEA, 1 microM)] or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 microM) followed by a 10 or 20 min washout or not preconditioned (control). Cardiomyocytes or whole hearts were then subjected to prolonged ischemic period (45 min simulated ischemia or 40 min of regional myocardial ischemia, respectively) followed by 60 min reperfusion (resuspension in oxygenated medium or release of the transient coronary occlusion, respectively). RESULTS: Indanyloxyacetic acid 94, a selective Cl(-) channel inhibitor that produced substantial inhibition of the regulatory volume decrease (RVD) when given at 10 microM concentration in cultured cardiomyocytes, was administered before ischemia to block RVD through Cl(-) channel inhibition. CCPA, APNEA and PMA significantly (P<0.01) reduced the % of dead cardiomyocytes (by trypan blue staining) after 45 min SI/60 min SR, as compared to controls, while IAA-94 abolished this protection but did not affect PKCepsilon translocation by IPC. We confirmed that IAA-94 blocked IPC-, APNEA- and PMA-induced protection against infarction in the isolated heart model. CONCLUSIONS: These findings support our contention that Cl(-) channels are downstream effectors of IPC.

Enhancement of natural killer cell activity in septic shock patients by a mixture of the calcium ionophore A23187 and the phorbol ester TPA: in vitro studies.

Preincubation of peripheral blood lymphocytes from drug-free, healthy volunteers with a mixture of the calcium ionophore A23187 (Io) and the phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) consistently resulted in a significant enhancement (dose-dependent; maximum immunostimulation obtained with the Io + TPA final mixture concentration of 10 uM + 250 ng/ml, respectively) of natural killer cell activity (NKCA) (n = 8; mean +/- SD of 16.8 +/- 8.9 and 52.0 +/- 18.0, paired Student's t-test p < 0.005; effector-to-target cell ratio of 30:1). Results from the same protocol, but using samples from septic shock patients followed a similar trend; however, and perhaps reflecting the significantly lower baseline NKCA in this group of individuals (n = 7), the mean value reached for this cellular immune function after incubation with Io + TPA was significantly lower than that of the treated controls' group (mean +/- SD of 19.8 +/- 11.6 and 52.0 +/- 18.0, respectively, Student's t-test p < 0.005). As expected from the role of calcium in the activation of NKCA, incubation with the Io significantly increased baseline NKCA, which was largely unchanged by TPA. Expression of the CD56+ and CD16+ phenotypes in septic shock patients did not correlate directly with NKCA, suggesting that this condition may be associated with changes in the function rather than the quantity of these cellular markers.(ABSTRACT TRUNCATED AT 250 WORDS)

[The role of protein kinase C in left ventricular relaxation impaired by global ischemia].

The role of protein kinase C (C kinase) in the left ventricular relaxation impaired by global ischemia was investigated in anesthetised dogs. Left ventricular global ischemia model was made by coronary blood flow reduction and atrial pacing (100-180 beats/min). By this maneuver, the time constant T and left ventricular end-diastolic pressure (LVEDP) were increased in a pacing-rate dependent manner. Intracoronary infusion of H-7, an inhibitor of C kinase, suppressed the magnitudes of the increments of T and LVEDP, while intracoronary infusion of 12-O-tetradecanoyl-phorbol-13-acetate, an activator of C kinase, enhanced the increases of T and LVEDP caused by ischemia. In non-ischemic group, H-7 did not influenced T and LVEDP. The results indicate that C kinase is activated by myocardial ischemia and enhances impairment of left ventricular relaxation.

12-O-Tetradecanoyl phorbol-13-acetate (TPA)-induced growth arrest is increased by silibinin by the down-regulation of cyclin B1 and cdc2 and the up-regulation of p21 expression in MDA-MB231 human breast cancer cells.

TPA is a potent regulator of cell growth, including cell proliferation and differentiation. In this study, we determined the effect of silibinin on TPA-induced growth arrest in breast cancer cells. Silibinin increased growth arrest of the G2/M phase in a dose-dependent fashion. Silibinin decreased the basal level of cyclin B1 and cdc2 expression, which is involved in S phase and G2/M transition. In addition, TPA-induced G2/M phase arrest was increased by silibinin. Under the same conditions, TPA-induced down-regulation of cyclin B1 and cdc2 was decreased by silibinin. In contrast, TPA-induced p21 expression was further increased by silibinin. To determine the regulatory mechanism of TPA-induced growth arrest, we pretreated cells with various inhibitors, such as UO126, SB203580, and LY294002. Interestingly, TPA-induced growth arrest was significantly increased by LY294002, but not by UO126 and SB203580. In addition, TPA-induced down-regulation of cyclin B1 was inhibited by LY294002; however, the basal level of p21 was increased by TPA and TPA-induced p21 expression was further increased by LY294002. Finally, adenoviral constitutively active-Akt (Ad-CA-Akt) overexpression regulated the up-regulation of cyclin B1 and the down-regulation of p21. Therefore, we have demonstrated that silibinin has an additive effect on TPA-induced growth arrest through the PI-3-kinase/Akt-dependent pathway.

Effect of intravenous infusions of 12-O-tetradecanoylphorbol-13-acetate (TPA) in patients with myelocytic leukemia: preliminary studies on therapeutic efficacy and toxicity.

Studies by several investigators have shown that 12-0-tetradecanoylphorbol-13-acetate (TPA) is an extraordinarily potent stimulator of differentiation of cultured human promyelocytic leukemia cells in vitro. In the present study, TPA was administered to humans by i.v. infusion without irreversible toxicity, and it was shown to have pharmacological activity for the treatment of myelocytic leukemia in patients refractory to cytosine arabinoside (Ara C), retinoic acid, and other antileukemic drugs. Marked decreases in bone marrow myeloblasts as well as temporary remission of disease symptoms were observed when TPA was administered alone or in combination with vitamin D3 and Ara C. Additional studies with TPA after the determination of optimum dosing regimens are needed to determine whether long-lasting or permanent remissions of myelocytic leukemia can be achieved. Transient and reversible side effects were observed after a 1-mg i.v. dose of TPA, but these adverse effects became less intense or disappeared when a lower dose of TPA was used. The results of this study indicate a therapeutic effect of TPA in patients with myelocytic leukemia.