Nelfinavir is effective against human cervical cancer cells in vivo: a potential treatment modality in resource-limited settings

Figures & data

Figure 1 Morphology of ME-180 cells with or without nelfinavir treatment.

Notes: Cells were treated with control 0.1% dimethyl sulfoxide or with 10 μM nelfinavir after 20 hours. While some proliferation is inhibited by nelfinavir by this time point, as indicated by lower cell confluence, the cellular morphology is relatively unchanged. ME-180 cells often have an obvious vacuole, which tends to increase in size following treatment with nelfinavir. The scale bar indicates 30 μm and applies to all images with the exception of the two magnified excerpts shown at the bottom of the figure.

Figure 2 Cervical cancer cells are sensitive to nelfinavir regardless of cisplatin sensitivity.

Notes: Parental ME-180 cells (ME-180) or daughter lines selected for growth in the presence of cisplatin (ME-180 CPR) were evaluated for the impact of 48-hour treatment with (A) cisplatin or (B) nelfinavir at the drug concentrations indicated. *P<0.05; ***P<0.001.
Abbreviation: CPR, cisplatin-resistant.

Figure 3 Nelfinavir induces autophagy.

Notes: (A) Western blot of lysates prepared following 24 hours of drug treatment. Nelfinavir was used at 10 μM. LC3-II indicates the autophagosomal form of LC3. Right panel quantifies LC3-II/actin ratio from three separate experiments using ImageJ. *P<0.05 by Wilcoxon rank-sum test. ns indicates P>0.05. (B) Immunofluorescence of cells treated with control 0.1% dimethyl sulfoxide or 10 μM nelfinavir for 24 hours. Scale bars indicate 10 μm and apply to all images. Punctate LC3 were quantified as autophagosomes and punctate p62 were quantified as sequestosomes, **P<0.01, ***P<0.001. (C and D) Western blot of lysates prepared following 24 hours of drug treatment. Nelfinavir was used at 10 μM and the autophagy clearance inhibitor chloroquine at 10 μM. (C) C7 represents caspase-7, and the cleaved form is associated with apoptosis. Grp78 is an endoplasmic reticulum stress marker. (D) RIPK1 cleavage and RIP3 expression are required for necroptosis, but increases compared to control were not observed in these experiments.
Abbreviations: CPR, cisplatin-resistant; fu, fluorescence units; ns, nonsignificant.

Figure 4 Nelfinavir suppresses cervical tumor growth in vivo.

Notes: ME-180 cells were injected into the left flank and ME-180 CPR cells were injected into the right flank of nude mice. Once the ME-180 CPR tumors reached a size of 100 mm³, the mice were begun on treatment, regardless of the size of the ME-180 tumors on the left flank. Treatment was by daily gavage, consisting of 50% PEG in water for control and 250 mg/kg nelfinavir in 50% PEG for nelfinavir treatment. After 21 days of treatment, the mice were euthanized. (A) Whole body weights of each group of mice throughout treatment; ns indicates P>0.05. (B) Volume of ME-180 or (C) ME-180 CPR tumors, as measured by calipers.
Abbreviations: CPR, cisplatin-resistant; ns, nonsignificant; PEG, polyethylene glycol.

Figure 5 Allelic suppression of core autophagy genes in cervical cancer.

Notes: The Cancer Genome Atlas was queried for allelic losses or gains of core autophagy genes. (A) ATG7 and ATG10 showed statistically significant enrichments for losses over gains; ***P<0.001 by Fisher’s exact test. (B) mRNA levels for ATG7 and ATG10 correlated with somatic copy number. (C) Allelic losses of these two core autophagy genes were tested for prognosis of patients. Only ATG10 loss conferred worse prognosis.
Abbreviations: mRNA, messenger RNA; RNAseq, RNA sequencing.