Ectopic expression of Protein kinase C-beta sensitizes head and neck squamous cell carcinoma to diterpene esters

Background: The objective of this study was to examine the effect of specific Protein kinase C (PKC) isoform re-expression in solid malignancies, particularly head and neck squamous cell carcinoma cell lines, and the impact this may have on treatment with known activators of PKC. Materials and Methods: The constitutive expression of PKC isoforms were determined in six head and neck squamous cell carcinoma (SCC) cell lines. Cytotoxicity of the prototypic phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the novel diterpene ester PEP005 was established. Viral transduction to re-express PKC beta isoforms in two of these cell lines was performed, and its effect on the sensitivity to the compounds was quantified. Results: Tongue and hypopharyngeal SCC cell lines were resistant to both TPA and PEP005, with the concentration required to inhibit growth by 50% (IC50) being >1,000 ng/ml. CAL-27 (tongue SCC) and FaDu (hypopharyngeal SCC) cell lines re-expressing PKC beta I and -beta II isoforms demonstrated IC50 of 1-5 ng/ml with TPA or PEP005. Conclusion: Re-expression of PKC beta in head and neck SCC cell lines leads to cells one thousand-times more sensitive to the cytotoxic effects of phorbol or diterpene esters in culture. This highlights the importance of the isoform in tumor progression and presents the potential benefit of these compounds in malignancies expressing the protein, and in combination therapy

Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models

<div><p>Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA <i>in vitro</i>, it was more effective for cure of tumors <i>in vivo</i>. No viable tumor cells were evident four hours after injection by <i>ex vivo</i> culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers <i>in vitro</i> and by CD31 immunostaining of treated tumors <i>in vivo</i>. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.</p></div

EBC-46 causes disruption and permeability of endothelial cells within tumor.

<p><b>A</b>. Immunohistochemical analysis of CD31 staining of FaDu head and neck cancer tumors treated with EBC-46. FaDu tumors were allowed to reach 100 mm³ before they were treated with either vehicle (20% propylene glycol in water) or 50 nmol (30 µg) EBC-46, and harvested at the indicated times. Representative photomicrographs are shown of the tumor site. Black arrows – examples of vessels with compromised or disrupted structural integrity. Scale bars  = 100 µm. <b>B</b>. Monolayers of HUVEC cells were treated with 350 µM (200 µg/ml) EBC-46 for 30 mins, before being assessed for permeability to FITC labeled Dextran. (***, p = 0.0013; t-test). <b>C</b>. HUVEC cells were treated with 350 µM (200 µg/ml) EBC-46 for 30 mins with or without 5 µM bisindolylmaleimide-1, before being assessed by propidium iodide exclusion. Error bars – SD, n = 3.</p

EBC-46 efficacy <i>in vivo</i> is independent of tumor cell sensitivity <i>in vitro</i>.

<p><b>A</b>. Dose response for cell killing by EBC-46 compared to PMA. B16-F0 (circles) or SK-MEL-28 (squares) melanoma cells were treated with the indicated doses of either EBC-46 (blue) or PMA (red) for 4 days, before assay for cell survival using the sulforhodamine B assay. Data shown are mean ± SD from triplicate readings from three independent experiments, n = 3. <b>B</b>. Kaplan Meier analysis of survival of C57BL/6J mice with B16-F0 tumors. Mice with two tumors were treated with single bolus doses of vehicle alone (20% propylene glycol in water; light grey), 50 nmol (30 µg) PMA (mid grey) or 50 nmol (30 µg) EBC-46 (black). Mice were euthanized once the total tumor volume reached 1,000 mm³ per animal. Squares – censored data. Difference between survival following treatment with EBC-46 or PMA was significant (*** p = 0.0004; Log-rank (Mantel-Cox) Test). Data was obtained from 6 mice per group, 2 tumors per mouse; n = 12. <b>C</b>. Kaplan Meier analysis of SK-MEL-28 melanoma tumors reaching >100 mm³ in BALB/c <i>Foxn1^nu</i> mice following single treatment with vehicle alone (grey) or 30 µg EBC-46 (black) (***, p<0.0001; Log-rank (Mantel-Cox) Test). Data was obtained from 5 mice per group, 2 tumors per mouse; n = 10. <b>D</b>. Kaplan Meier analysis of MM649 melanoma tumors reaching >100 mm³ in BALB/c <i>Foxn1^nu</i> mice following single treatment with vehicle alone (grey) or 50 nmol (30 µg) EBC-46 (black) (***, p<0.0001; Log-rank (Mantel-Cox) Test). Data was obtained from 5 mice per group, 2 tumors per mouse; n = 10.</p

EBC-46 treatment induces greater effects when injected into tumors compared to normal skin.

<p>EBC-46 was injected into male BALB/c <i>Foxn1^nu</i> mice either bearing (n = 9) or not bearing (n = 9) tumors, as a single intra-lesional or sub-cutaneous injection, respectively. <b>A</b>. Erythema at injection site 24 hours following treatment with 50 nmol (30 µg) EBC-46 in tumor- or non-tumor-bearing mice. <b>B</b>. Oedema at injection site 24 hours following treatment with 50 nmol (30 µg) EBC-46 in tumor- or non-tumor-bearing mice. <b>C</b>. Percentage weight change 24 hours following treatment with 50 nmol (30 µg) EBC-46 in tumor- or non-tumor-bearing mice. <b>D</b>. Concentration in serum following treatment with 50 nmol (30 µg) EBC-46 in tumor- or non-tumor-bearing mice. Data is from serum from three animals unless otherwise indicated. Error bars – SEM. (**, p<0.01; ***, p<0.005; t-test). a - single data point from EBC-46-treated animals. b - below lower limit of detection of the assay, set at 0.01 ng/ml.</p

Clinical scoring for Drug Treated and Tumor Bearing Mice.

<p>Clinical scoring for Drug Treated and Tumor Bearing Mice.</p

EBC-46 anti-cancer efficacy is PKC-dependent.

<p><b>A</b>. The effect of neutrophil depletion on EBC-46 treatment of SK-MEL-28 tumors grown on BALB/c <i>Foxn1^nu</i> mice. Tumor growth in mice given anti-Ly-6G antibody, isotype control antibody or no antibody. SK-MEL-28 cells were injected sub-cutaneously (n = 10 tumors/group; 2 tumors/mouse, 5 mice/group) and allowed to reach approximately 100 mm³. Mice were injected with anti-Ly-6G antibody (clone 1A8; 100 µg i.p, on days −2, 0, and 2), with isotype control antibody (IgG2a, clone 2A3; 100 µg i.p, on days −2, 0, and 2) or with nothing. Tumors were treated by intra-lesional injection of vehicle (50 µl of 20% propylene glycol in water) on day 0. The tumor volumes represent the mean volume of individual tumors. • – no antibody, ▪ – control IgG2a antibody, ▴ – anti-mLy-6G antibody. <b>B</b>. Tumor growth in mice given anti-Ly-6G antibody, isotype control antibody or no antibody after EBC-46 treatment. As for A, SK-MEL-28 tumors (approximately 100 mm³) were treated by intra-lesional injection with 25 nmol (15 µg) EBC-46 (in 50 µl of 20% propylene glycol in water) on day 0. • – no antibody, ▪ – control IgG2a antibody, ▴ – anti-mLy-6G antibody. Error bars – SD. <b>C</b>. 500,000 B16-F0 cells were injected into BALB/c <i>Foxn1^nu</i> mice, and allowed to reach >50 mm³. Tumors were then treated with either 50 µl vehicle (20% propylene glycol in water), 16.7 nmol (10 µg) EBC-46 in vehicle or 16.7 nmol (10 µg) EBC-46 after pre-treatment with 5 µM bisindolylmaleimide-1 (BIS-1) in vehicle. Tumor size was measured 8 days after treatment. Error bars  =  SD, n = 12. (****, p<0.0001; **, p = 0.0024; t-test). <b>D</b>. Number of tumors (as percentage) present 8 days after treatment. Total tumor number n = 12 for each group.</p