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Kalkitoxin inhibits angiogenesis, disrupts cellular hypoxic signaling, and blocks mitochondrial electron transport in tumor cells.

Morgan JB, Liu Y, Coothankandaswamy V, Mahdi F, Jekabsons MB, Gerwick WH, Valeriote FA, Zhou YD, Nagle DG - Mar Drugs (2015)

Bottom Line: Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM).Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase).Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of BioMolecular Sciences and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA. jbmorga1@go.olemiss.edu.

ABSTRACT
The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-D-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells.

No MeSH data available.


Related in: MedlinePlus

Kalkitoxin suppresses tumor cell proliferation/viability in a cell line- and time-dependent manner. (A) Exponentially grown T47D, MDA-MB-231, and SH-SY5Y cells were exposed to kalkitoxin at the specified concentrations for 48 h (2 days) and 144 h (6 days), respectively. Cell viability was determined by the SRB method and presented as “% Inhibition” of the untreated control (average ± standard deviation, n = 3); (B) HCT116 cells were exposed to kalkitoxin at the specified concentrations for 5 days and the number of surviving cells determined by trypan blue exclusion. Surviving fraction data are presented as the average ± standard deviation (n = 3); (C) Following kalkitoxin treatment for 2, 24, and 168 h at the specified concentrations, HCT116 cells were detached and plated at low density. Seven days later, the number of colonies was counted and the surviving fraction data presented (average ± standard deviation, n = 3).
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marinedrugs-13-01552-f006: Kalkitoxin suppresses tumor cell proliferation/viability in a cell line- and time-dependent manner. (A) Exponentially grown T47D, MDA-MB-231, and SH-SY5Y cells were exposed to kalkitoxin at the specified concentrations for 48 h (2 days) and 144 h (6 days), respectively. Cell viability was determined by the SRB method and presented as “% Inhibition” of the untreated control (average ± standard deviation, n = 3); (B) HCT116 cells were exposed to kalkitoxin at the specified concentrations for 5 days and the number of surviving cells determined by trypan blue exclusion. Surviving fraction data are presented as the average ± standard deviation (n = 3); (C) Following kalkitoxin treatment for 2, 24, and 168 h at the specified concentrations, HCT116 cells were detached and plated at low density. Seven days later, the number of colonies was counted and the surviving fraction data presented (average ± standard deviation, n = 3).

Mentions: In general, standard cytostatic/cytotoxic assays of 48 h duration are performed to evaluate the anticancer potential of active leads [31]. Our studies and those of McLaughlin and coworkers’ indicate that an extended exposure time (e.g., six days) is required to observe the full impact of mitochondrial ETC inhibitors on tumor cell proliferation and/or viability [28,32]. To determine the effects of kalkitoxin on tumor cell proliferation/viability, concentration-response studies were performed following both standard and extended exposure schedule (48 h and 144 h, respectively). Enhanced inhibition was observed with all three-cell lines (human breast cancer T47D and MDA-MB-231, and neuroblastoma SH-SY5Y) in the extended exposure study (Figure 6A). The most pronounced increase was observed in T47D cells (Figure 6A). Additionally, exponentially grown HCT116 cells were exposed to kalkitoxin at the specified concentrations for five days and the surviving cells monitored using the trypan blue excluding method. Kalkitoxin decreased HCT116 survival with an IC50 value of 1 ng/mL (or 2.7 nM, Figure 6B). The impact of kalkitoxin on tumor cell survival was further examined in a clonogenic assay. HCT116 cells were exposed to kalkitoxin for the specified amount of time (2 h, 24 h, and 168 h, respectively), and the ability of treated cells to form colonies determined (Figure 6C). While little if any cell killing occurred for either a 2 or 24 h exposure for concentrations up to 100 µg/mL (270 µM), an extended exposure (168 h) to kalkitoxin was required to significantly suppress tumor cell colony formation, similar to that observed in Figure 6B.


Kalkitoxin inhibits angiogenesis, disrupts cellular hypoxic signaling, and blocks mitochondrial electron transport in tumor cells.

Morgan JB, Liu Y, Coothankandaswamy V, Mahdi F, Jekabsons MB, Gerwick WH, Valeriote FA, Zhou YD, Nagle DG - Mar Drugs (2015)

Kalkitoxin suppresses tumor cell proliferation/viability in a cell line- and time-dependent manner. (A) Exponentially grown T47D, MDA-MB-231, and SH-SY5Y cells were exposed to kalkitoxin at the specified concentrations for 48 h (2 days) and 144 h (6 days), respectively. Cell viability was determined by the SRB method and presented as “% Inhibition” of the untreated control (average ± standard deviation, n = 3); (B) HCT116 cells were exposed to kalkitoxin at the specified concentrations for 5 days and the number of surviving cells determined by trypan blue exclusion. Surviving fraction data are presented as the average ± standard deviation (n = 3); (C) Following kalkitoxin treatment for 2, 24, and 168 h at the specified concentrations, HCT116 cells were detached and plated at low density. Seven days later, the number of colonies was counted and the surviving fraction data presented (average ± standard deviation, n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4377999&req=5

marinedrugs-13-01552-f006: Kalkitoxin suppresses tumor cell proliferation/viability in a cell line- and time-dependent manner. (A) Exponentially grown T47D, MDA-MB-231, and SH-SY5Y cells were exposed to kalkitoxin at the specified concentrations for 48 h (2 days) and 144 h (6 days), respectively. Cell viability was determined by the SRB method and presented as “% Inhibition” of the untreated control (average ± standard deviation, n = 3); (B) HCT116 cells were exposed to kalkitoxin at the specified concentrations for 5 days and the number of surviving cells determined by trypan blue exclusion. Surviving fraction data are presented as the average ± standard deviation (n = 3); (C) Following kalkitoxin treatment for 2, 24, and 168 h at the specified concentrations, HCT116 cells were detached and plated at low density. Seven days later, the number of colonies was counted and the surviving fraction data presented (average ± standard deviation, n = 3).
Mentions: In general, standard cytostatic/cytotoxic assays of 48 h duration are performed to evaluate the anticancer potential of active leads [31]. Our studies and those of McLaughlin and coworkers’ indicate that an extended exposure time (e.g., six days) is required to observe the full impact of mitochondrial ETC inhibitors on tumor cell proliferation and/or viability [28,32]. To determine the effects of kalkitoxin on tumor cell proliferation/viability, concentration-response studies were performed following both standard and extended exposure schedule (48 h and 144 h, respectively). Enhanced inhibition was observed with all three-cell lines (human breast cancer T47D and MDA-MB-231, and neuroblastoma SH-SY5Y) in the extended exposure study (Figure 6A). The most pronounced increase was observed in T47D cells (Figure 6A). Additionally, exponentially grown HCT116 cells were exposed to kalkitoxin at the specified concentrations for five days and the surviving cells monitored using the trypan blue excluding method. Kalkitoxin decreased HCT116 survival with an IC50 value of 1 ng/mL (or 2.7 nM, Figure 6B). The impact of kalkitoxin on tumor cell survival was further examined in a clonogenic assay. HCT116 cells were exposed to kalkitoxin for the specified amount of time (2 h, 24 h, and 168 h, respectively), and the ability of treated cells to form colonies determined (Figure 6C). While little if any cell killing occurred for either a 2 or 24 h exposure for concentrations up to 100 µg/mL (270 µM), an extended exposure (168 h) to kalkitoxin was required to significantly suppress tumor cell colony formation, similar to that observed in Figure 6B.

Bottom Line: Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM).Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase).Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells.

View Article: PubMed Central - PubMed

Affiliation: Department of BioMolecular Sciences and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA. jbmorga1@go.olemiss.edu.

ABSTRACT
The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-D-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells.

No MeSH data available.


Related in: MedlinePlus