Lycopene modulates cellular proliferation, glycolysis and hepatic ultrastructure during hepatocellular carcinoma

Table 1 List of primer pairs used

Gene	Strand	Primer
HIF-1α	Sense	5’-GGT/CAG/ATG/ATC/AGA/GTC/C-3’
	Antisense	5’-TGC/TTG/GTG/CTG/ATT/TGTG/A-3’
PCNA	Sense	5’-GAT/GTG/GAG/CAA/CTT/GGA/AT-3’
	Antisense	5’-AGC/TCT/CCA/ACT/TGC/AGA/AAA-3’
p21	Sense	5’-CCG/TGG/ACA/GTG/AGC/AGT/TG -3’
	Antisense	5’-TGG/GCA/CTT/CAG/GGT/TTT/CT-3’
Cyclin D1	Sense	5’-CAC/AAC/GCA/CTT/TCT/TTC/CA-3’
	Antisense	5’-GAC/CAG/CCT/CTT/CCT/CCA/C-3’
β-actin	Sense	5’-ATC/CGT/AAA/GAC/CTC/TAT/GC-3’
	Antisense	5’-AAC/GCA/GCT/CAG/TAA/CAG/TC-3’

Table 2 Comparative analysis of the N-Nitrosodiethylamine and lycopene extracted from tomatoes + N-Nitrosodiethylamine group by scanning electron microscopy

Groups/parameters	NDEA	LycT + NDEA
Cell density	+++	+
Rounding of hepatocytes	+++	+
Trabecular structures	-	+++
Necrotic tumor nodules	+++	+
Apoptotic bodies	-	+++
Type of HCC	Undifferentiated	Well-differentiated

Where “+++” indicates that more than 70%-90% of mice in a group showed this feature; “+” indicates that < 70% of mice showed this feature; “-” indicates that the < 20% of mice showed this feature. LycT: Lycopene extracted from tomatoes; NDEA: N-Nitrosodiethylamine; HCC: Hepatocellular carcinoma.

Table 3 Comparative analysis of the N-Nitrosodiethylamine and lycopene extracted from tomatoes + N-Nitrosodiethylamine group by transmission electron microscopy

Groups/parameters	NDEA	LycT + NDEA
Hepatocytes	Rounded, smaller in size	Polygonal but rounded edges
Nucleus	Large and irregular shape	Oval shaped
Nuclear membrane	Not uniform and pseudoinclusions	Not uniform
Nucleoli	Large, irregular, multiple	One-two
Nuclear/cytoplasmic ratio	High	Low
Karyotin (reticular material)	Deposition along nuclear membrane	Fewer karyotin deposits
Cytoplasm	Loss of organization, dense	Organized
Mitochondria	Pleomorphic with increased density	Pleomorphic but number less than that in the NDEA group
Lysosomes	Few in number	High in number
Fat and glycogen globules	Variable	High
Macrophages	Very few	Many
Apoptotic bodies	No	Clearly visible in the section

LycT: Lycopene extracted from tomatoes; NDEA: N-Nitrosodiethylamine.

Table 4 Effect of N-Nitrosodiethylamine and/or lycopene extracted from tomatoes on protein expression in mice hepatic tissue using ELISA

Protein (absorbance at 405 nm)	Control	NDEA	LycT	LycT + NDEA
PCNA	0.31 ± 0.01	0.47 ± 0.01a	0.31 ± 0.01b	0.39 ± 0.01a,b,c
Cyclin D1	0.25 ± 0.02	0.38 ± 0.02a	0.24 ± 0.02b	0.36 ± 0.01a,c
p21	0.40 ±0.02	0.33 ± 0.02e	0.39 ± 0.02f	0.37 ± 0.01g
HIF-1α	0.22 ± 0.03	0.41 ± 0.01a	0.23 ± 0.03b	0.36 ± 0.04a,c

^aP ≤ 0.05, compared to the NDEA group;

^b,c,eP ≤ 0.001, compared to the control group, NDEA group and LycT group, respectively;

^f,gP ≤ 0.01, compared to the control group and NDEA group, respectively. LycT: Lycopene extracted from tomatoes; NDEA: N-Nitrosodiethylamine; PCNA: Proliferating cell nuclear antigen; p21: Cyclin-dependent kinase inhibitor 1A; HIF-1α: Hypoxia inducible factor-1α.