Combination Of Gossypol Research Articles

Gossypol synergises antiproliferative effect of sorafenib in metastatic lung cancer cells following Chou-Talalay algorithm

Combination therapy has been proposed as a promising approach for lung cancer treatment, as it can enhance anticancer efficacy, and reduce dosages and adverse effects. This study aimed to explore the therapeutic potential of gossypol, a natural polyphenolic compound with sorafenib for treating lung cancer cells and elucidating its mechanism of action. The MTT assay was utilized to determine the IC50 of sorafenib and gossypol against A549 and NCI H460 cell lines. The Chou-Talaly algorithm was employed to determine the combination index (CI). A sub-effective concentration of sorafenib and gossypol was chosen to investigate the possibility of cytotoxic synergy. Autophagy biomarkers were identified using Western blotting, and the function of autophagy was determined using ATG5 siRNA. Results show that IC50 of sorafenib significantly reduced in A549 and NCI H460 cells when co-treated with gossypol. The combination treatment showed a synergistic cytotoxic effect against tested cell lines. The Chou-Talaly algorithm confirmed sorafenib's dose reduction index (DRI) up to 3.86. In A549 cells, combination treatment down-regulated p62 and up-regulated LC3-II, indicating the initiation of autophagy-dependent cytotoxicity. This was further confirmed by siRNA ATG5 knockdown. Additionally, the combination treatment exclusively targeted G0/G1 phase cancer cells. In conclusion, the combination of gossypol and sorafenib shows a synergistic increase in the cytotoxic effect by promoting autophagy and apoptosis.

Combinatorial Therapeutic Effect of Inhibitors of Aldehyde Dehydrogenase and Mitochondrial Complex I, and the Chemotherapeutic Drug, Temozolomide against Glioblastoma Tumorspheres.

Resident cancer cells with stem cell-like features induce drug tolerance, facilitating survival of glioblastoma (GBM). We previously showed that strategies targeting tumor bioenergetics present a novel emerging avenue for treatment of GBM. The objective of this study was to enhance the therapeutic effects of dual inhibition of tumor bioenergetics by combination of gossypol, an aldehyde dehydrogenase inhibitor, and phenformin, a biguanide compound that depletes oxidative phosphorylation, with the chemotherapeutic drug, temozolomide (TMZ), to block proliferation, stemness, and invasiveness of GBM tumorspheres (TSs). Combination therapy with gossypol, phenformin, and TMZ induced a significant reduction in ATP levels, cell viability, stemness, and invasiveness compared to TMZ monotherapy and dual therapy with gossypol and phenformin. Analysis of differentially expressed genes revealed up-regulation of genes involved in programmed cell death, autophagy, and protein metabolism and down-regulation of those associated with cell metabolism, cycle, and adhesion. Combination of TMZ with dual inhibitors of tumor bioenergetics may, therefore, present an effective strategy against GBM by enhancing therapeutic effects through multiple mechanisms of action.

Targeting Protein Neddylation to Inactivate Cullin-RING Ligases by Gossypol: A Lucky Hit or a New Start?

Cullin-RING E3 ligases (CRLs) are the largest family of E3 ubiquitin ligases, responsible for about 20% of the protein degradation by the ubiquitin-proteasome system (UPS). Given their vital roles in multiple cellular processes, and over-activation in many human cancers, CRLs are validated as promising targets for anti-cancer therapies. Activation of CRLs requires cullin neddylation, a process catalysed by three neddylation enzymes. Recently, our group established an AlphaScreen-based in vitro cullin neddylation assay and employed it for high-throughput screening to search for small-molecule inhibitors targeting cullin neddylation. During our pilot screen, gossypol, a natural product extracted from cottonseeds, was identified as one of the most potent neddylation inhibitors of cullin-1 and cullin-5. We further demonstrated that gossypol blocks cullin neddylation by binding to cullin-1/-5 to inactivate CRL1/5 ligase activity, leading to accumulation of MCL-1 and NOXA, the substrates of CRL1 and CRL5, respectively. The combination of gossypol and an MCL-1 inhibitor synergistically enhanced the anti-proliferative effect in multiple human cancer cell lines. Our study unveiled a rational combination of two previously known inhibitors of the Bcl-2 family for enhanced anti-cancer efficacy and identified a novel activity of gossypol as an inhibitor of CRL1 and CRL5 E3s, thus providing a new possibility in the development of novel CRL inhibitors for anti-cancer therapy.

Polyphenol‐Based Nanomedicine Evokes Immune Activation for Combination Cancer Treatment

AbstractEngineering multifunctional nanoplatforms with high therapeutic benefits has become a promising strategy for intractable cancer treatment. A novel polyphenol‐based nanocomplex was designed to evoke highly efficacious cancer immunosurveillance while localizing therapy on the primary tumor and to minimize systemic side effects. This nanocomplex is prepared via metal–polyphenol coordination by encapsulating a natural polyphenol, gossypol, and a newly synthesized polyphenol derivative, polyethylene glycol‐Chlorin e6 (Ce6). The combination of gossypol from cotton and the photosensitizer Ce6 can induce chemotherapeutic/photodynamic immunogenic cancer cell death upon laser irradiation, which is supported by a rich maturation of dendritic cells, concentrated secretion of inflammatory cytokines, and significant inhibition of distant untreated tumors. Finally, an assistance of the programmed‐cell‐death ligand‐1 checkpoint‐blockade immunotherapy can enhance the anti‐tumor immune stimulation of our nanoplatform to a higher level.

Polyphenol-Based Nanomedicine Evokes Immune Activation for Combination Cancer Treatment.

Engineering multifunctional nanoplatforms with high therapeutic benefits has become a promising strategy for intractable cancer treatment. A novel polyphenol-based nanocomplex was designed to evoke highly efficacious cancer immunosurveillance while localizing therapy on the primary tumor and to minimize systemic side effects. This nanocomplex is prepared via metal-polyphenol coordination by encapsulating a natural polyphenol, gossypol, and a newly synthesized polyphenol derivative, polyethylene glycol-Chlorin e6 (Ce6). The combination of gossypol from cotton and the photosensitizer Ce6 can induce chemotherapeutic/photodynamic immunogenic cancer cell death upon laser irradiation, which is supported by a rich maturation of dendritic cells, concentrated secretion of inflammatory cytokines, and significant inhibition of distant untreated tumors. Finally, an assistance of the programmed-cell-death ligand-1 checkpoint-blockade immunotherapy can enhance the anti-tumor immune stimulation of our nanoplatform to a higher level.

Gossypol inhibits cullin neddylation by targeting SAG-CUL5 and RBX1-CUL1 complexes

Cullin-RING E3 ligase (CRL) is the largest family of E3 ubiquitin ligase, responsible for ubiquitylation of ∼20% of cellular proteins. CRL plays an important role in many biological processes, particularly in cancers due to abnormal activation. CRL activation requires neddylation, an enzymatic cascade transferring small ubiquitin-like protein NEDD8 to a conserved lysine residue on cullin proteins. Recent studies have validated that neddylation is an attractive anticancer target. In this study, we report the establishment of an Alpha-Screen-based high throughput screen (HTS) assay for in vitro CUL5 neddylation, and screened a library of 17,000 compounds including FDA approved drugs, natural products and synthetic drug-like small-molecule compounds. Gossypol, a natural compound derived from cotton seed, was identified as an inhibitor of cullin neddylation. Biochemical studies showed that gossypol blocked neddylation of both CUL5 and CUL1 through direct binding to SAG-CUL5 or RBX1-CUL1 complex, and CUL5-H572 plays a key role for gossypol binding. On cellular level, gossypol inhibited cullin neddylation in a variety of cancer cell lines and selectively caused accumulation of NOXA and MCL1, the substrates of CUL5 and CUL1, respectively, in multiple cancer cell lines. Combination of gossypol with specific MCL1 inhibitor synergistically suppress growth of human cancer cells. Our study revealed a previously unknown anti-cancer mechanism of gossypol with potential to develop a new class of neddylation inhibitors.

Identification of Novel Natural Products as Effective and Broad-Spectrum Anti-Zika Virus Inhibitors.

Zika virus (ZIKV) infection during pregnancy leads to severe congenital Zika syndrome, which includes microcephaly and other neurological malformations. No therapeutic agents have, so far, been approved for the treatment of ZIKV infection in humans; as such, there is a need for a continuous effort to develop effective and safe antiviral drugs to treat ZIKV-caused diseases. After screening a natural product library, we have herein identified four natural products with anti-ZIKV activity in Vero E6 cells, including gossypol, curcumin, digitonin, and conessine. Except for curcumin, the other three natural products have not been reported before to have anti-ZIKV activity. Among them, gossypol exhibited the strongest inhibitory activity against almost all 10 ZIKV strains tested, including six recent epidemic human strains. The mechanistic study indicated that gossypol could neutralize ZIKV infection by targeting the envelope protein domain III (EDIII) of ZIKV. In contrast, the other natural products inhibited ZIKV infection by targeting the host cell or cell-associated entry and replication stages of ZIKV. A combination of gossypol with any of the three natural products identified in this study, as well as with bortezomib, a previously reported anti-ZIKV compound, exhibited significant combinatorial inhibitory effects against three ZIKV human strains tested. Importantly, gossypol also demonstrated marked potency against all four serotypes of dengue virus (DENV) human strains in vitro. Taken together, this study indicates the potential for further development of these natural products, particularly gossypol, as the lead compound or broad-spectrum inhibitors against ZIKV and other flaviviruses, such as DENV.

Gossypol Suppresses Growth of Temozolomide-Resistant Glioblastoma Tumor Spheres.

Temozolomide is the current first-line treatment for glioblastoma patients but, because many patients are resistant to it, there is an urgent need to develop antitumor agents to treat temozolomide-resistant glioblastoma. Gossypol, a natural polyphenolic compound, has been studied as a monotherapy or combination therapy for the treatment of glioblastoma. The combination of gossypol and temozolomide has been shown to inhibit glioblastoma, but it is not clear yet whether gossypol alone can suppress temozolomide-resistant glioblastoma. We find that gossypol suppresses the growth of temozolomide-resistant glioblastoma cells in both tumor sphere and adherent culture conditions, with tumor spheres showing the greatest sensitivity. Molecular docking and binding energy calculations show that gossypol has a similar affinity to the Bcl2 (B-cell lymphoma 2) family of proteins and several dehydrogenases. Gossypol reduces mitochondrial membrane potential and cellular ATP levels before cell death, which suggests that gossypol inhibits several dehydrogenases in the cell’s metabolic pathway. Treatment with a Bcl2 inhibitor does not fully explain the effect of gossypol on glioblastoma. Overall, this study demonstrates that gossypol can suppress temozolomide-resistant glioblastoma and will be helpful for the refinement of gossypol treatments by elucidating some of the molecular mechanisms of gossypol in glioblastoma.

Targeting Mitochondrial Oxidative Phosphorylation Abrogated Irinotecan Resistance in NSCLC

Anticancer drug resistance is a major challenge of cancer therapy. We found that irinotecan-resistant NSCLC cells showed increased mitochondrial oxidative phosphorylation compared to the drug sensitive NSCLC cells. Previously, we found that combined inhibition of aldehyde dehydrogenase using gossypol, and mitochondrial complex I using phenformin, effectively reduced oxidative phosphorylation in NSCLC. Here, we showed that targeting oxidative phosphorylation with gossypol and phenformin abrogated irinotecan resistance in NSCLC. Furthermore, irinotecan treatment by blocking oxidative phosphorylation induced synergistic anti-cancer effect in NSCLC. The pre-clinical xenograft model of human NSCLC also demonstrated a therapeutic response to the dual targeting treatment. Therefore, this combination of gossypol and phenformin increases irinotecan sensitivity as well as preventing irinotecan resistance.

Examination of Gossypol-Pluronic Micelles as Potential Radiosensitizers.

Chemoradiotherapy, the combination of chemotherapy and radiotherapy to treat cancer, has the potential to enhance local therapeutic effects and simultaneously treat systemic disease. However, chemoradiotherapy may also enhance normal tissue effects leading to both acute and late toxicities. Furthermore, subtherapeutic chemoradiotherapy may result in aggressive tumor repopulation. Tumor-specific radiosensitizing chemotherapy may yield a synergistic therapeutic effect and avoid augmentation of normal tissue toxicity. In this study, the radiosensitizing effects of gossypol were investigated. Also, Pluronics were studied for gossypol solubilization and co-radiosensitization effects. Gossypol inhibits Bcl-2 and Bcl-XL, antiapoptotic proteins that are overexpressed in various cancer cells. Pluronic micelles (P85, F88, L35, and P123) effectively encapsulated gossypol, raising its water solubility by more than 1000-fold. Cytotoxic, anticlonogenic, and radiosensitizing effects were evaluated to characterize gossypol and Pluronic combinations. Gossypol and P85 had the strongest antiproliferative effect on A549 human lung adenocarcinoma cells in a cell viability assay. The IC50 value was seven times lower than gossypol only treatment (330 ± 70nM vs 2400 ± 400nM, (mean ± SE)). Gossypol and P85 showed significant inhibition of clonogenic survival, approximately 30% inhibition, compared to treatment with gossypol alone. An experimental sequencing study demonstrated greater inhibition of clonogenic survival when drug treatment followed radiation compared to a sequence of drug treatment followed by radiation. These results suggest that Pluronic micelles readily solubilize gossypol and that the combination of gossypol and P85 may augment the therapeutic effects of ionizing radiation.

Gossypol and an HMT G9a inhibitor act in synergy to induce cell death in pancreatic cancer cells

The histone methyltransferase G9a is overexpressed in a variety of cancer types, including pancreatic adenocarcinoma, and promotes tumor invasiveness and metastasis. We recently reported the discovery of BRD4770, a small-molecule inhibitor of G9a that induces senescence in PANC-1 cells. We observed that the cytotoxic effects of BRD4770 were dependent on genetic background, with cell lines lacking functional p53 being relatively resistant to compound treatment. To understand the mechanism of genetic selectivity, we used two complementary screening approaches to identify enhancers of BRD4770. The natural product and putative BH3 mimetic gossypol enhanced the cytotoxicity of BRD4770 in a synergistic manner in p53-mutant PANC-1 cells but not in immortalized non-tumorigenic pancreatic cells. The combination of gossypol and BRD4770 increased LC3-II levels and the autophagosome number in PANC-1 cells, and the compound combination appears to act in a BNIP3 (B-cell lymphoma 2 19-kDa interacting protein)-dependent manner, suggesting that these compounds act together to induce autophagy-related cell death in pancreatic cancer cells.

The role of COX‐2/PGE2 in gossypol‐induced apoptosis of colorectal carcinoma cells

Our previous study showed that gossypol (GOS) exhibits potent cytotoxic effects via apoptosis induction against human colorectal carcinoma cells; however, the role of cyclooxygenase (COX)-2/prostaglandin (PG)E(2) on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma HT-29 cells in accordance with inducing COX-2 protein/PGE(2) production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase (PK)C inhibitors including staurosporine (ST), GF109203X (GF), and H7, characterized by the occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE(2) production in HT-29 cells. The addition of COX activity inhibitors, including NS398 (NS), aspirin (AS), diclofenac (DI), and indomethacin (IN), suppressed TPA protection of GOS-induced apoptosis with decreased PGE(2) production in HT-29 cells. Application of PGE(2), but not it analogs PGD(2), PGJ(2), or PGF(2α), protected HT-29 cells from GOS-induced DNA ladders, and the E-prostanoid (EP(1)) receptor agonist, 17PT-PGE(2), mimicked the protection induced by PGE(2), whereas the selective EP(2) receptor agonist, butaprostol (BUT), the EP(3) receptor agonist, sulprostol (SUL), and the EP(4) receptor agonist, PGE(1) alcohol (PGE(1)), showed no significant effects on GOS-induced apoptosis in HT-29 cells. PGE(2) 's protection against GOS-induced apoptosis was reversed by adding the selective EP(1) receptor antagonist, SC-19220. Furthermore, GOS had an effective apoptotic effect on COLO205 colorectal carcinoma cells which expressed undetectable level of endogenous COX-2 protein than HT-29 cells, and the decreased COX-2 protein level via COX-2 siRNA or addition of COX-2 activity inhibitor NS significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection or enhanced GOS-induced cell death according to MTT assay and DNA integrity assay. The notion of COX-2/PGE(2) activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and the combination of GOS and COX-2 inhibitors to treat colon carcinoma possesses clinical potential worthy of further investigation.

Abstract C34: The role of COX-2/PGE2 in gossypol-induced apoptosis of colorectal carcinoma cells

Abstract Our previous study has shown that gossypol (GOS) exhibited the potent cytotoxic effect via apoptosis induction against human colorectal carcinoma cells; however, the role of cyclooxygenase 2 (COX-2)/prostaglandin E2 on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma cells COLO205 in according with inducing COX-2 protein/PGE2 production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase C (PKC) inhibitors including ST, GF, and H7, characterized by an occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE2 production in COLO205 cells. Addition of COX activity inhibitors including NS398, aspirin (AS), diclofenac (DI), and indomethacin (IN) suppressed TPA protection of GOS-induced apoptosis with decreased PGE2 production in COLO205 cells. Application of PGE2, but not it analogues PGD2, PGJ2, and PGF2, protected COLO205 cells from GOS-induced DNA ladders, and the EP1 receptor agonist 17PT-PGE2 mimicked the protection by PGE2, whereas the selective EP2 receptor agonist butaprostol (BUT), EP3 receptor agonist sulprostol (SUL), and EP4 receptor agonist PGE1 alcohol (PGE1) showed no significant effect on GOS-induced apoptosis in COLO205 cells. PGE2 protection of GOS-induced apoptosis was reversed by adding a selective EP1 receptor antagonist SC-19220. Furthermore, GOS had less apoptotic effect on HT-29 colorectal carcinoma cells which expressed higher level of endogenous COX-2 protein than COLO205 cells, and decreased COX-2 protein level via COX-2 siRNA significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection enhanced GOS-induced cell death by MTT assay. The notion of COX-2/PGE2 activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and combination of GOS and COX-2 inhibitors to treat colon carcinoma reserved clinical potential for further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C34.

Anti‐amoebic Effect of Gossypol in Golden Hamsters with Experimental Hepatic Amoebic Abscess

Although metronidazole is an effective anti-amoebic medication, it is mutagenic and some strains of Entamoeba histolytica resistant to this drug have begun to appear. The search for new anti-amoebic compounds is, therefore, justified. We have previously found that gossypol has a potent in-vitro anti-amoebic effect; we now show that this compound is also effective in-vivo. Hamsters with experimental amoebic hepatic abscess were fed acetic acid gossypol (0–45 mg kg−1) or metronidazole (30 or 45 mg kg−1) for 5 or 10 days. The experimental amoebic hepatic abscess size and the in-vitro cell density reached by trophozoite cultures from the experimental amoebic hepatic abscesses were scored. Gossypol and metronidazole reduced the experimental amoebic hepatic abscess. The smallest effect of gossypol was obtained with 5–45 mg kg−1 (23–31% score reduction) administered for 5 days whereas the most effective treatment corresponded to 30 mg kg−1 gossypol for 10 days (90% score reduction). Gossypol at 45 mg kg−1 for 10 days was toxic. The anti-amoebic effect of 30 mg kg−1, judged as the cell density score reduction compared with the control group, was comparable with that produced by 30 mg kg−1 metronidazole administered for 10 days. Although in molar concentration terms, gossypol was nine times more potent than metronidazole, the margin between the most effective and lethal doses of gossypol was relatively narrow. A combination of gossypol and metronidazole could be a more efficacious and less toxic anti-amoebic combination than the separate drugs.

Gossypol exerts its cytotoxic effect on HL-60 leukemic cell line via decreasing activity of protein phosphatase 2A and interacting with human telomerase reverse transcriptase activity

In this study, we aimed to investigate the potential relationship between gossypol-induced cytotoxicity of human promyelocytic leukemia cell line (HL-60) leukemic cells and intracellular serine/threonine protein phosphatase (PP) dynamics and human telomerase reverse transcriptase (hTERT) activity. Gossypol was found to be cytotoxic in HL-60 cells with the IC50 dose of 4·5 μM. The combination of gossypol and okadaic acid in IC50 doses revealed the increased cytotoxicity in a time-dependent manner. Treatment of cells with gossypol has shown significant decrease in PP2A activity. The expression of the PP2A catalytic subunit was downregulated in gossypol-treated cells with 24 hours' intervals. hTERT mRNA levels were gradually decreased. In conclusion, during gossypol-induced cytotoxicity, intracellular activity and expression of PP2A was decreased as well as the activity of hTERT. The variation of hTERT activity in gossypol-treated HL-60 cells may be the potential reason for the phosphatase interaction during the gossypol treatment of leukemic cells resulting in cellular cytotoxicity.

Gossypol Activates Pancreatic Polyamine Catabolism in Normal Rats and Induces Acute Pancreatitis in Transgenic Rats Over-expressing Spermidine/Spermine N 1 -Acetyltransferase

Background: The male antifertility agent gossypol has been reported to induce spermidine/spermine N 1 -acetyltransferase (SSAT) in canine prostate cells. As SSAT is the rate-controlling enzyme in the catabolism of the polyamines and is involved in the development of acute pancreatitis in a recent transgenic rat model, we exposed normal and transgenic rats over-expressing SSAT to gossypol to evaluate its effect on pancreatic polyamine metabolism and organ integrity. Methods: Pancreatic SSAT activity, polyamine pools, pancreatic histology and plasma ! -amylase activity were determined after different doses of gossypol. Results: Gossypol increased pancreatic putrescine and decreased spermidine and spermine pools in normal rats accompanied by tissue oedema and significantly elevated plasma amylase activity. In transgenic rats, the drug strikingly induced SSAT, profoundly depleted the higher polyamines and caused distinct pancreatitis. The combination of gossypol at doses harmless to transgenic pancreas with an inhibitor of polyamine oxidase caused massive synergistic induction of SSAT, profound depletion of the polyamine pools and acute pancreatitis. Conclusions: The results indicate that gossypol induces pancreatitis through an activation of polyamine catabolism.

Modification of the positive inotropic effects of catecholamines, cardiac glycosides and Ca 2+ by the orally active male contraceptive, gossypol, in isolated guinea-pig heart

Gossypol is an orally active male contraceptive with cardio-depressant side effects. To understand the mechanism of its cardiac actions, the interaction of gossypol with positive inotropic grugs was examined in isolated atrial muscle preparations obtained from guinea- pig heart. Gossypol delayed the onset of arrhythmias caused by digoxin. In the presence of gossypol, the positive inotropic effect of isoproterenol declined rapidly, and the effect of isoproterenol to increase tissue cyclic AMP concentrations was smaller. Pretreatment of atrial muscle with the combination of gossypol and isoproterenol markedly reduced effects of isoproterenol on developed tension and cyclic AMP concentrations when these effects were tested after the washout of the first dose of isoproterenol. These effects, however, were not specific to isoproterenol. The gossypol-isoproterenol pretreatment reduced the positive inotropic effect of ouabain or extracellular Ca 2+ These results indicate that gossypol has pharmacodynamic interactions with several positive inotropic agents that are known to enhance developed tension by increasing intracellular Ca 2+ transients.